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Florida International UniversityFIU Digital Commons
FIU Electronic Theses and Dissertations University Graduate School
6-17-2015
Design and Synthesis of Novel NucleosideAnalogues: Oxidative and Reductive Approachestoward Synthesis of 2'-Fluoro PyrimidineNucleosidesRamanjaneyulu RayalaFlorida International University, rraya002@fiu.edu
DOI: 10.25148/etd.FIDC000129Follow this and additional works at: https://digitalcommons.fiu.edu/etd
Part of the Carbohydrates Commons, Heterocyclic Compounds Commons, and the NucleicAcids, Nucleotides, and Nucleosides Commons
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Recommended CitationRayala, Ramanjaneyulu, "Design and Synthesis of Novel Nucleoside Analogues: Oxidative and Reductive Approaches towardSynthesis of 2'-Fluoro Pyrimidine Nucleosides" (2015). FIU Electronic Theses and Dissertations. 2172.https://digitalcommons.fiu.edu/etd/2172
FLORIDA INTERNATIONAL UNIVERSITY
Miami, Florida
DESIGN AND SYNTHESIS OF NOVEL NUCLEOSIDE ANALOGUES: OXIDATIVE
AND REDUCTIVE APPROACHES TOWARD SYNTHESIS OF 2'-FLUORO
PYRIMIDINE NUCLEOSIDES
A dissertation submitted in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
in
CHEMISTRY
by
Ramanjaneyulu Rayala
2015
ii
To: Dean Michael R. Heithaus College of Arts and Sciences
This dissertation, written by Ramanjaneyulu Rayala, and entitled Design and Synthesis of Novel Nucleoside Analogues: Oxidative and Reductive Approaches toward Synthesis of 2'-Fluoro Pyrimidine Nucleosides, having been approved in respect to style and intellectual content, is referred to you for judgment.
We have read this dissertation and recommend that it be approved.
_______________________________________ Kathleen S. Rein
_______________________________________
David A. Becker
_______________________________________ Anthony P. DeCaprio
_______________________________________
Salvatore D. Lepore
_______________________________________ Stanislaw F. Wnuk, Major Professor
Date of Defense: June 17, 2015
The dissertation of Ramanjaneyulu Rayala is approved.
_______________________________________ Dean Michael R. Heithaus
College of Arts and Sciences
_______________________________________ Dean Lakshmi N. Reddi
University Graduate School
Florida International University, 2015
iii
DEDICATION
This Ph.D. is dedicated to:
Padma Rayala, Nagaiah Rayala, Aneela Rayala, Yashitha Rayala, Radhika Mandapalli,
Muralidhar Mandapalli, Rishitha Chowdary Mandapalli, and Srikarprasad Chowdary
Mandapalli.
You have been a great inspiration.
iv
ACKNOWLEDGMENTS
I would like to thank my mentor Professor Stanislaw F. Wnuk for giving me the
opportunity to work in his research group and for his constant support over the years.
Also, I am really thankful to my dissertation committee members for their constructive
criticism and guidance throughout my PhD. I also want to thank senior group members
Jean-Philippe Pitteloud, Adam Sobczak, and Aparna Malladi for their guidance during
my initial days in the lab. I would like to give many special thanks to my friends at FIU
Jesse Pulido, Jessica Zayas, Yong Liang, Mukesh Mudgal, Nagarju Birudukota, Cesar
Gonzalez, and Sazzad Hossain for their support and tolerance, they have been an
extended family to me. I would like to acknowledge my undergraduate students Patricia
Theard, Brenna Walsh and Maria Barrios for assisting me during different stages of my
dissertation.
I would like to express my sincere gratitude to the following people and
institutions who supported me and provided me with guidance at various phases of my
life: Ravi Convent, Ms. Mamatha, Naveen and all the wonderful people of Vuyyalawada,
Dornakal; Mr. Venkateshwarlu, and other faculty and staff at Sri Vivekananda
Vidyalayam, Dornakal; Faculty and staff at Spectra Junior College, Khammam; Faculty
and staff at DRS Degree College, Khammam; Faculty and staff at PG College of Science,
Saifabad; Faculty and staff at KLR Degree College, Palwancha; Mr. Harikrishna and Ms.
Bhuwaneshwari, and other faculty and staff at VISU, Hyderabad; Mr. Verma and Ms.
Padma at Periscope, Hyderabad; Seniors from my M.Sc., Ramesh, Raghu, and Satheesh;
My friends Hareesh, Srikanth, Anil, Harish, Sudhakar, Vijay, Santosh, Surya, Alankar,
Madhukar, and Chandu.
v
I want to show my gratitude to the Department of Chemistry and Biochemistry at
Florida International University for providing me with Teaching Assistantship for major
part of my PhD. I would like to thank the University Graduate School for the Dissertation
Year Fellowship Award which was immensely helpful during my last year as a graduate
student. Last, but not the least, I would like to thank Florida International University and
the United States of America.
vi
ABSTRACT OF THE DISSERTATION
DESIGN AND SYNTHESIS OF NOVEL NUCLEOSIDE ANALOGUES: OXIDATIVE
AND REDUCTIVE APPROACHES TOWARD SYNTHESIS OF 2'-FLUORO
PYRIMIDINE NUCLEOSIDES
by
Ramanjaneyulu Rayala
Florida International University, 2015
Miami, Florida
Professor Stanislaw F. Wnuk, Major Professor
Fluorinated nucleosides, especially the analogues with fluorine atom(s) in the
ribose ring, have been known to exert potent biological activities. The first part of this
dissertation was aimed at developing oxidative desulfurization-fluorination and reductive
desulfonylation-fluorination methodologies toward the synthesis of 2'-mono and/or 2',2'-
difluoro pyrimidine nucleosides from the corresponding 2'-arylthiopyrimidine precursors.
Novel oxidative desulfurization-difluorination methodology was developed for the
synthesis of α,α-difluorinted esters from the corresponding α-arylthio esters, wherein the
arylthio group is present on a secondary internal carbon. For the reductive
desulfonylation studies, cyclic voltammetry was utilized to measure the reduction
potentials at which the sulfone moiety of substrates can be cleaved.
The 5-bromo pyrimidine nucleosides and 8-bromo purine nucleosides act as
crucial intermediates in various synthetic transformations. The second part of the present
dissertation was designed to develop a novel bromination methodology using 1,3-
dibromo-5,5-dimethylhydantoin (DBH). Various protected and deprotected pyrimidine
vii
and purine nucleosides were converted to their respective C5 and C8 brominated
counterparts using DBH. The effect of Lewis acids, solvents, and temperature on the
efficiency of bromination was studied. Also, N-bromosuccinimide (NBS) or DBH offered
a convenient access to 8-bromotoyocamycin and 8-bromosangivamycin.
Third part of this research work focuses on the design and synthesis of 6-N-
benzylated derivatives of 7-deazapurine nucleoside antibiotics, such as tubercidin,
sangivamycin and toyocamycin. Target molecules were synthesized by two methods.
First method involves treatment of 7-deazapurine substrates with benzylbromide
followed by dimethylamine-promoted Dimroth rearrangement. The second method
employs fluoro-diazotization followed by SNAr displacement of the 6-fluoro group by a
benzylamine. The 6-N-benzylated 7-deazapurine nucleosides showed type-specific
inhibition of cancer cell proliferation at micromolar concentrations and weak inhibition
of human equilibrative nucleoside transport protein (hENT1).
In the fourth part of this dissertation, syntheses of C7 or C8 modified 7-
deazapurine nucleosides, which might exhibit fluorescent properties, were undertaken. 8-
Azidotoyocamycin was synthesized by treatment of 8-bromotoyocamycin with sodium
azide. Strain promoted click chemistry of 8-azidotoyocamycin with cyclooctynes gave
the corresponding 8-triazolyl derivatives. Alternatively, 7-benzotriazolyl tubercidin was
synthesized by iodine catalyzed CH arylation of tubercidin with benzotriazole.
viii
TABLE OF CONTENTS
CHAPTER PAGE 1. INTRODUCTION .......................................................................................................... 1
1.1. Notable nucleoside analogues with anticancer and antiviral activity ................... 2 1.2. Importance of fluorine substitution ...................................................................... 3 1.3. Prominent nucleoside analogues with fluorine atom in sugar moiety .................. 4 1.4. Gemcitabine, a critical anticancer drug ................................................................ 5 1.4.1. Existing methods for the synthesis of gemcitabine ........................................... 5 1.4.2. Mechanism of action of gemcitabine ................................................................ 7 1.5. Short survey on desulfurization-fluorination reactions ...................................... 10 1.6. 5-Bromopyrimidines and 8-bromopurines: Syntheses and importance ............. 12 1.6.1. 1,3-dibromo-5,5-dimethylhydantoin, an important brominating reagent ........ 13 1.7. Selected aspects of the chemistry of 7-deazapurine nucleosides ....................... 14 1.8. Nucleoside transport inhibitors .......................................................................... 16
2. RESEARCH OBJECTIVES ......................................................................................... 17 3. RESULTS AND DISCUSSION ................................................................................... 22
3.1. Bromination of nucleobases with 1,3-dibromo-5,5-dimethylhydantoin ............ 22 3.1.1 Bromination at C5 position of pyrimidine nucleosides with DBH .................. 22 3.1.2 Bromination at C8 position of purine nucleosides with DBH .......................... 28 3.1.3. Bromination at C8 position of 7-deazapurine nucleosides with NBS and
DBH ............................................................................................................. 31 3.1.4. Plausible mechanism of bromination of nucleobases with DBH .................... 33 3.2. Attempted synthesis of 2'-deoxy-2',2''-difluoropyrimidine nucleosides ............ 35 3.2.1. Oxidative desulfurization-fluorination approaches ......................................... 36 3.2.1.1. Model desulfurization-fluorination studies .................................................. 36 3.2.1.2. Oxidative desulfurization-fluorination approaches with uridine derivatives 43 3.2.2. Reductive desulfonylation-fluorination approaches ........................................ 47 3.2.2.1. Cyclic voltammetry studies of 2'-arylsulfonyl uridine derivatives .............. 48 3.2.2.2. Reductive desulfonylation-fluorination studies of 2'-deoxy-2'-arylsulfonyl
uridine and 2'-deoxy-2'-fluoro-2'-arylsulfonyl uridine substrates ................ 51 3.3. One electron oxidation of gemcitabine and analogues ....................................... 59 3.3.1. Detection of C2' and C3' sugar radicals .......................................................... 60 3.4. Synthesis and biological activity of 6-N-benzyl-7-deazapurine nucleoside
derivatives .......................................................................................................... 62 3.4.1. Synthesis of 6-N-benzyl-7-deazapurine nucleosides ....................................... 62 3.4.2. Biological activity of 6-N-benzyl-7-deazapurine nucleoside derivatives ....... 68 3.4.2.1. Nucleoside transport inhibition activity ....................................................... 68 3.4.2.2. Antiviral activity ........................................................................................... 69 3.4.2.3. Inhibition of Cell Proliferation ..................................................................... 70 3.5. C7- and C8-modified-7-deazapurine nucleoside derivatives ............................. 71
ix
3.5.1. Strain promoted click reactions of 8-azido-7-deazanucleosides with cyclooctynes ................................................................................................. 71
3.5.1. Attempted synthesis of 8-alkynyl-7-deazanucleosides via Sonagashira reaction ......................................................................................................... 74
3.5.2. Iodine catalyzed direct C-H activation of 7-deazapurine nucleosides ............ 79
4. EXPERIMENTAL SECTION ...................................................................................... 82
5. CONCLUSION ........................................................................................................... 124
REFERENCES ............................................................................................................... 127
x
LIST OF TABLES
TABLE PAGE
Table 1. FDA approved anticancer purine and pyrimidine nucleoside analogues. ............ 2
Table 2. Antiviral properties of purine and pyrimidine nucleoside analogues. .................. 3
Table 3. Effect of various reaction parameters on C5-bromination of 2',3',5'-tri-O-acetyluridine 45a with DBHa ............................................................................. 24
Table 4. Bromination at C5 position of the pyrimidine based nucleosides 45a-f and 57-59a ................................................................................................................. 26
Table 5. Bromination of selected purine nucleosides at ambient temperature (see Figure 11 for structures)a ................................................................................... 30
Table 6. Desulfurization-difluorination studies of α-thioester derivatives with DBH/Py.9HFa .................................................................................................... 38
Table 7. Antiviral activity in human embryonic lung (HEL) cell cultures-part 1 ............ 69
Table 8. Antiviral activity human embryonic lung (HEL) cell cultures-part 2 ................ 70
Table 9. Inhibitory effects on the proliferation of cells in culture .................................... 71
Table 10. Reaction conditions for the attempted Sonagashira cross-coupling reactions on 8-bromo tubercidin and toyocamycina ........................................................ 76
xi
LIST OF FIGURES
FIGURE PAGE
Figure 1. Structures of RNA and DNA nucleosides ........................................................... 1
Figure 2. Examples of the prominent nucleoside drugs with fluorine modifications in sugar moieties. .................................................................................................. 4
Figure 3. Original synthesis of gemcitabine developed by Hertel. ..................................... 5
Figure 4. Synthesis of 2'-difluorocytidine derivatives from 2'-keto derivatives by deoxyfluorination .............................................................................................. 6
Figure 5. Synthesis of 2'-deoxy-2'-2'-difluorouridine derivative by desulfurization-difluorination of the corrssponding 2'-dithioketal derivative ........................... 7
Figure 6. Gemcitabine cellular metabolism. ....................................................................... 9
Figure 7. Mechanism for the covalent inhibition of RNR by dFdCDP in the absence and in the presence of reductant, proposed by Prof. Stubbe, et al. ................. 10
Figure 8. Structures of DBH and NBS.............................................................................. 14
Figure 9. Structures of 7-deazapurine nucleosides ........................................................... 14
Figure 10. Important base-modified 7-deazapurine nucleoside derivatives. .................... 15
Figure 11. Structures of Important nucleoside transport inhibitors. ................................ 17
Figure 12. Different classes of 2'-arylthionucleosides for oxidative desulfurization-fluorination and reductive desulfonylation-fluorination procedures. ............. 18
Figure 13. Various aryl-alkyl thioether substrates for model oxidative desulfurization-difluorination reactions ................................................................................... 19
Figure 14. Gemcitabine and its analogues for one electron oxidation studies.................. 20
Figure 15. Structures of 7-deazapurine antibiotics and the proposed 6-N-benzyl analogues......................................................................................................... 21
Figure 16. Structures of various 8-modified-7-deazapurine derivatives .......................... 21
Figure 17. Selected pyrimidine nucleoside precursors (series a) and their brominated products (series b). .......................................................................................... 28
Figure 18. Selected purine nucleoside precursors (series a) and their brominated products (series b). .......................................................................................... 29
xii
Figure 19. General scheme for the proposed C2'-quarternization of 2'-arylthiouridine derivatives ....................................................................................................... 36
Figure 20. α-Thioaldehyde, α-thioketone and α-thioalkane substrates for attempted desulfurization-(di)fluorination reactions. ...................................................... 41
Figure 21. Products for attempted fluorination of 2'-S-(4-chlorophenyl)-2'-thiouridine derivatives 89a and 89b .................................................................................. 46
Figure 22. Cyclic voltammetry of α-sulfones 92a (C = 2.25 mM, blue curve) and 92b (C = 2.75 mM, pink curve); in DMF + n-Bu4NPF6 0.1M; v = 0.2 V/s. .......... 49
Figure 23. Cyclic voltammetry of α-fluorosulfones 95a (2.64 mM) and 95b (2.91 mM) 50
Figure 24. Structures of organic electron donors used for the reductive desulfonylation. 53
Figure 25. Selected substrates for 1e- oxidation studies ................................................... 60
xiii
LIST OF SCHEMES
SCHEME PAGE
Scheme 1. Oxidative desulfurization-difluorination of aldehyde, ketone dithiolanes; and alkyl aryl thioethers. ............................................................................... 11
Scheme 2. Hara's oxidative desulfurization-difluorination of benzylic sulfides. ............. 12
Scheme 3. Synthesis of 4-N-methyltubercidin by Dimroth-rearrangement approach ...... 16
Scheme 4. Bromination of uracil-derived nucleosides 1 with 1,3-dibromo-5,5-dimethylhydantoin (DBH). See Table 3 and 4 for specific reaction parameters. .................................................................................................... 23
Scheme 5. Synthesis of 8-bromo-7-deazanucleosides. Reagents and conditions: d) NBS (1.1-1.5 eq), DMF, rt, 1-2.5 h; e) DBH (0.75-1.1 eq), MeOH, rt, 30-45 min. ..................................................................................................... 32
Scheme 6. Plausible mechanism of C-5 bromination of pyrimidine nucleosides by DBH. .............................................................................................................. 33
Scheme 7. Plausible mechanism of C-8 bromination of purine nucleosides by DBH. .... 34
Scheme 8. Plausible mechanism of TMSOTf enhanced DBH bromination. .................... 35
Scheme 9. General scheme for Haufe’s desulfurization-difluorination of aryl-alkyl thioethers. ...................................................................................................... 36
Scheme 10. Desulfurization-difluorination of α-thioesters. .............................................. 37
Scheme 11. Proposed mechanism for the oxidative desulfurization-difluorination of alkyl-aryl thioethers by DBH/Py.9HF ........................................................... 40
Scheme 12. Attempted synthesis of α,α-difluoro-δ-valerolactone 82. ............................. 42
Scheme 13. Attempted synthesis of α,α-difluoro-γ-butyrolactone 86. ............................. 42
Scheme 14. Synthesis of 2'-arylthio substrates for the desulfurization-fluorination reactions. ........................................................................................................ 44
Scheme 15. Oxidative desulfurization-difluorination of 2'-S-aryl-2'-thiouridine analogue 89a. Conditions A: DBH (1.1 eq), Py.9HF (excess), CH2Cl2, -78 oC, 2 h; Conditions B: NIS (3 eq), DAST (6 eq), CH2Cl2, 0 oC (6 h), then rt (32 h); Conditions C: NIS (3 eq), DAST (6 eq), CH2Cl2, 35 oC, 24 h. ............................................................................................................... 45
xiv
Scheme 16. Attempted oxidative desulfurization-fluorination of 2'-S-(4-methoxyphenyl)-2'-thiouridine analogue 90b. Conditions A: NIS (2.1 eq), Py.9HF (3 eq); Conditions B: NIS (2.1 eq), DAST (6 eq). ..... 47
Scheme 17. Attempted oxidative desulfurization-fluorination of 2'-fluoro-2'-S-(4-methoxyphenyl)-2'-thiouridine analogue 94b with DBH/Py.9HF. ............... 47
Scheme 18. Stability studies of sulfones 91a-b on silica gel chromatography or with treatment of base. .......................................................................................... 51
Scheme 19. Reduction studies of acetyl protected sulfone substrates 91a with TDAE and Murphy’s SED. ....................................................................................... 54
Scheme 20. Reduction studies of benzyl protected sulfone substrate 91b with Murphy’s SED. .............................................................................................. 55
Scheme 21. Stability studies of acetyl protected α-fluorosulfone 95a with Murphy’s SED. .............................................................................................................. 56
Scheme 22. Stability studies of benzyl protected α-fluorosulfones 95b with Murphy’s SED ............................................................................................................... 57
Scheme 23. Attempted synthesis of 2'-2''-difluorouridine by reductive desulfonylation-fluorination of 2'-arylsulfonyl-2'-deoxy-2'-fluorouridine 95a with TDAE/Selectrfluor ........................................................................................ 58
Scheme 24. Reductive-desulfonylation/methylation of 2'-arylsulfonyl-2'-deoxy-2'-fluorouridine 95a with TDAE/MeI ................................................................ 59
Scheme 25. One elctron oxidation of 2'-deoxy-2'-fluorocytidine ..................................... 61
Scheme 26. One electron oxidation of gemcitabine ......................................................... 61
Scheme 27. One electron oxidation of PSI-6130 .............................................................. 62
Scheme 28. General mechanism of Dimroth rearrangement on Adenosine183,184 ............ 63
Scheme 29. Synthesis of 4-N-benzylated tubercidin and sangivamycin by Dimroth rearrangement approach. ............................................................................... 64
Scheme 30. Synthesis of 4-N-(4-nitrobenzyl)toyocamycin by direct alkylation. ............. 65
Scheme 31. Synthesis of 2',3',5'-tri-O-acetyl-4-fluorotubercidin (124) by diazotive-fluorodeamination. ........................................................................................ 67
Scheme 32. Synthesis of 4-N-(4-nitrobenzyl)tubercidin (122b) from 2',3',5'-tri-O-acetyl-4-fluorotubercidin (124). ................................................ 68
xv
Scheme 33. Synthesis of 8-bromo-7-deazaadenosine and 8-azidotoyocamycin derivatives. .................................................................................................... 73
Scheme 34. Click reaction between 8-azidotoyocamycin and cyclooctynes 130 and 131. Reagents and conditions: g) Cyclooctyne 130 or 131 (1 eq), ACN:H2O (3:1), rt, 4 h. ................................................................................. 74
Scheme 35. Proposed scheme for the synthesis and subsequent transformations of 8-alkynyl-7-deazapurine nucleoside analogues 135. ........................................ 75
Scheme 36. Attempted synthesis of 8-alkynyltoyocamycin derivative 140 via sonagashira coupling. .................................................................................... 78
Scheme 37. Attempted synthesis of 8-alkynyltoyocamycin derivative 143 via sonagashira coupling. .................................................................................... 79
Scheme 38. Iodine catalyzed oxidative cross-coupling of Indoles and Azoles. ............... 79
Scheme 39. Iodine catalyzed oxidative cross-coupling of tubercidin and benzotrizole. .. 80
Scheme 40. Iodine catalyzed oxidative cross-coupling of acetylated tubercidin and benzotriazole. ................................................................................................ 81
Scheme 41. Attempted synthesis of iodine catalyzed oxidative cross-coupling of sangivamycin or toyocamycin and benzotriazole. ........................................ 82
xvi
LIST OF ABBREVIATIONS
Ac acetyl
Ar aromatic (NMR)
β beta
Bn benzyl
br broad (NMR)
t-But tert-butyl
calcd calculated (MS or HRMS)
CDA cytidine deaminase
º C degrees Celsius
δ delta
d doublet (NMR) or deuterium (in DMSO-d6)
DAST diethylaminosulfur trifluoride
dCK deoxycytidine kinase
DCM dichloromethane
DCTD deoxycytidylate deaminase
dFdC 2',2'-difluoro-2'-deoxycytidine
dFdCMP 2',2'-difluoro-2'-deoxycytidine monophosphate
dFdCDP 2',2'-difluoro-2'-deoxycytidine diphosphate
dFdCTP 2',2'-difluoro-2'-deoxycytidine triphosphate
DMAP 4-(N,N-dimethylamino)pyridine
DMF N,N-dimethylformamide
DMSO dimethylsulfoxide
xvii
DNA deoxyribonucleic acid
dUMP deoxyuridine monophosphate
ε epsilon
ESI electrospray ionization
Et ethyl
FdC 2'-fluoro-2'-deoxycytidine
g gram(s)
h or hr or hrs hour(s)
Hz hertz
hCNT human concentrative nucleoside transporter
hENT human equilibrative nucleoside transporter
HPLC high performance liquid chromatography
HRMS high resolution mass spectroscopy
IC50 half maximal inhibitory concentration
J coupling constant in Hz (NMR)
λ lambda
L liter(s)
LDA lithium diisopropylamide
m milli; multiplet (NMR)
µ micro
M moles per liter
MeFdC 2'-deoxy-2'-fluoro-2'-C-methylcytidine
min minute(s)
xviii
mol mole(s)
MS mass spectrometry
m/z mass to charge ratio (MS)
NMR nuclear magnetic resonance
nM nano Molar
p para
PET Positron Emission Tomography
pyr pyridine
% percentage
q quartet (NMR)
quin quintet (NMR)
RNA ribonucleic acid
RNR Ribonucleotide reductase
rt room temperature
RP reverse phase (HPLC)
s second(s); singlet (NMR)
SET single electron transfer
t triplet (NMR)
TBDMS tert-butyldimethylsilyl
TBDPS tert-butyldiphenylsilyl
TBAF tetra-n-butylammonium fluoride
TBHP tert-butyl hydroperoxide
TEA triethylamine
xix
THF tetrahydrofuran
TLC thin layer chromatography
TMS trimethylsilyl
tR retention time (HPLC)
Tris tris(hydroxymethyl)aminomethane
UV ultraviolet
VIS visible
vs versus
1
1. INTRODUCTION
In 1909, the term “nucleoside” was proposed by Levene and Jacobs to describe
carbohydrate derivatives of purines and pyrimidines.1 Nucleosides, held together by
phosphodiester linkages, are the core components of the biopolymers DNA and RNA.
Nucleosides are composed of a furanose sugar moiety, linked to either a purine or a
pyrimidine heterocyclic base via a beta-glycosidic bond. The four nucleosides prevailing
in RNA are adenosine, guanosine, cytosine and uridine. Whereas, 2'-deoxyadenosine, 2'-
deoxyguanosine, 2'-deoxycytidine and thymidine form the core part in DNA (see Figure
1 for structures of RNA and DNA nucleosides). Development of drug therapy progressed
rapidly after the discovery of DNA as the primary genetic material in 19442 and
subsequent elucidation of its physical structure in 1953.3 Since then, DNA and RNA have
been regarded as potential targets for drug design, as they play vital role in many
biological processes. Since nucleosides form the major part of DNA and RNA,
nucleosides have also been considered prospective targets.
Figure 1. Structures of RNA and DNA nucleosides3
2
1.1. Notable nucleoside analogues with anticancer and antiviral activity
Pyrimidine nucleoside analogues such as 5-fluoro-2'-deoxyuridine (Floxuridine),
1-(β-ᴅ-arabinofuranosyl)cytosine (araC) were among the initial nucleoside analogues
approved by U.S. Food and Drug Administration (FDA) for cancer treatment (Table 1).
Remarkable biological activity resulting from slight structural modifications to the
normal nucleosides prompted various scientists all over the world to develop modified
nucleoside analogues as anticancer agents. Currently, there are 11 nucleoside based drugs
approved by FDA for the treatment of cancer.
Table 1. FDA approved anticancer purine and pyrimidine nucleoside analogues.4
Drug Category Year
5-aza-2'-deoxycytidine (Decitabine) 2′-Deoxycytidine 2006
O6-methylarabinofuranosyl guanine (Nelarabine) Guanosine 2005
5-aza-cytidine (Vidaza) 2′-Deoxyadenosine 2004
2'-fluoro-2'-deoxyarabinofuranosyl-2-chloroadenine (Clofarabine) Cytidine 2004
N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine (Capecitabine) Cytidine 1998
2,2-difluoro-2'-deoxycytidine (Gemcitabine) 2′-Deoxycytidine 1996
2-chloro-2'-deoxyadenosine (Cladribine) 2′-Deoxyadenosine 1992
2'-deoxycoformycin (Pentostatin) Adenosine 1991
Arabinofuranosyl-2-fluoroadenine (Fludarabine) Purine analogue 1991
5-fluoro-2'-deoxyuridine (Floxuridine) 2′-Deoxyuridine 1970
Arabinofuranosylcytosine (Cytarabine) Cytidine 1969
6-thioguanine (Lanvis) Guanine 1966
5-fluorouracil (Adrucil) Uracil 1962
6-mercaptopurine (Purinethol) Purine 1953
Parallel to the anticancer drugs, researchers also developed various nucleoside
analogues with antiviral properties. The sounding success of prominent antiviral drugs
such as Acyclovir (anti-HSV), and Zidovudine (anti-HIV) revealed the potential value of
3
modified nucleoside analogues as antiviral agents (Table 2). There are currently 13 FDA
approved nucleoside based drugs used in the treatment of various viruses. Most recent of
them being Sofosbuvir, an anti-HCV drug, which is considered to be a huge step forward
in HCV treatment.5,6
Table 2. Antiviral properties of purine and pyrimidine nucleoside analogues.4
Drug Category Year 2'-deoxy-2'-fluoro-2'-methyluridine-5'-phosphate (Sofosbuvir) Uridine 2013
Tenofovir disoproxil fumarate, mixture of purine analogues (Tenofovir) Purine 2008
1-2-deoxy-β-L-erythro-pentofuranosylthymine (Telbivudine) Thymidine 2006
5-fluoro-1-(2R,5S)-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (Emtricitabine) Cytidine 2003
2',3'-didehydro-2',3'-dideoxythymidine (Stavudine) Thymidine 2001
2',3'-dideoxy-3'-thiacytidine (Lamivudine) Cytidine 1998
2',3'-didehydro-2',3'-dideoxythymidine (Stavudine) Thymidine 1994
3'-azido-3'-deoxythymidine (Zidovudine) Thymidine 1987
2-amino-1,9-dihydro-9-[(2-hydroxyethoxy)methyl]-6H-purin-6-one (Acyclovir) Guanosine 1982
(E)-5-(2-bromovinyl)-2'-deoxyuridine (Brivudine) Uridine 1980
9-β-D-arabinofuranosyladenine (Vidarabine) Adenosine 1976
5-ethyl-2'-deoxyuridine (Edoxudine) Thymidine 1969
5-iodo-2'-deoxyuridine (Idoxuridine) Uridine 1962
1.2. Importance of fluorine substitution
Fluoro organic compounds have been known to possess a wide variety of
biological activity and several of them are used as drugs for the treatment of various
types of diseases.7,8 Introduction of fluorine atom(s) into the organic molecules
dramatically changes their electronic and steric properties, which often leads to the
molecules rendering potent biological activities.9,10 Though a fluorine atom is close in
size to a hydrogen atom, presence of fluorine causes various electronic changes because
of its electronegativity. Moreover, fluorine atoms can also act as hydrogen bond
4
acceptors. Also, a fluorine atom can serve as a replacement for hydrogen, since the Van
der Waals radius of fluorine (1.35 Ǻ) is so close to that of a hydrogen atom (1.1 Ǻ). The
C-F bond strength (484 kJ/mol) is also greater than that of a C-H bond (411 kJ/mol),
offering a measure of increased biological and chemical stability to the structure.
1.3. Prominent nucleoside analogues with fluorine atom in sugar moiety
Of the variety of nucleoside analogues with biological activity, the class of fluoro
nucleosides is known to possess a wide range of medicinal and biological activity.11
Prominent examples of fluorinated nucleoside analogues include: 2'-deoxy-2',2'-
difluorocytidine (1, Gemcitabine)12,13 the phospharamidate analogue of 2'-deoxy-2'-
fluoro-2'-C-methyluridine (2, Sofosbuvir) and 2-chloro-1-(2'-deoxy-2'-
fluoroarabinofuranosyl)uracil (3, Clofarabine)14,15 (Figure 2). Gemcitabine (1) and
Clofarabine (3) are potent anti-cancer drugs that are commonly used in the treatment of
various types of cancers.13,16 Sofosbuvir (3) acts as potent inhibitor of Hepatitis C virus
replication.5,6
Figure 2. Examples of the prominent nucleoside drugs with fluorine modifications in sugar moieties.
1
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Over the last 25 years, several research groups have published improved methods
for the synthesis of gemcitabine. Some of these synthetic approaches followed Hertel’s
original approach and differ mainly in employing either different protecting groups on the
ribose or different coupling conditions to improve stereoselectivity/isomer-separation.
Additionally gemcitabine was also synthesized from various furanose and pyranose
derivatives. The synthesis of gemcitabine has been subject to an excellent review
published recently.21
Even though the synthesis of gemcitabine is widely studied from the stand point
of sugar-base coupling, much less attention has been paid toward the synthesis from
parent nucleosides. To our knowledge, only three literature examples have described the
synthesis of gemcitabine via direct carbohydrate fluorination of the parent nucleoside
derivatives.
A 1996 patent from Eli Lily reported the synthesis of 2'-deoxy-2',2'-
difluorocytidine derivative 6 by treating the corresponding 2'-keto cytidine derivative 5
with DAST and Pyr.HF reagent combination (Figure 4). Whereas a more recent Chinese
patent (2013) described the similar transformation with the new deoxyfluorinating agents
Xtalfluor-M and Xtalfluor-E.
Figure 4. Synthesis of 2'-difluorocytidine derivatives from 2'-keto derivatives by deoxyfluorination
7
Synthesis of difluorouridine derivative 10 by desulfurization-difluorination of the
corresponding dithiouridine derivative 9 with DBH/Py.9HF reagent combination was
reported in a 2007 patent (Figure 5). However, the crucial uridine-2'-dithioketal precursor
9, was prepared by coupling the uracil base with the pre-constructed dithioacetal ribose
derivative 8, which in turn was prepared from the open chain ribonolactone derivative
7.22
Figure 5. Synthesis of 2'-deoxy-2'-2'-difluorouridine derivative by desulfurization-difluorination of the corrssponding 2'-dithioketal derivative
1.4.2. Mechanism of action of gemcitabine
The mechanism of action of gemcitabine has been widely studied13,16-20 and is
only discussed briefly here. Gemcitabine (dFdC) is a prodrug and must be metabolized to
the active triphosphate form 2′,2′-difluoro-2′-deoxycytidine triphosphate (dFdCTP) to
exert biological activity (Figure 6). Human nucleoside transporters (hNTs), an important
class of membrane proteins, mediate the cellular uptake of gemcitabine. Gemcitabine is
converted into its monophosphate (dFdCMP) by deoxycytidine kinase (dCK); to its
diphosphate (dFdCDP) by pyrimidine nucleoside monophosphate kinase (UMP-CMP
kinase); and subsequently to the active metabolite triphosphate by nucleoside diphosphate
8
kinase. Gemcitabine may become inactivated through deamination by cytidine deaminase
(CDA) and, when in the monophosphate form by deoxycytidylate deaminase (dCTD).
The product of gemcitabine deamination by CDA is 2′,2′-difluoro-2′-deoxyuridine
(dFdU), which is cytotoxic itself. Also, dFdU monophosphate (dFdUMP) could inhibit
the activity of thymidylate synthase, directly effecting the deoxynucleotide triphosphate
(dNTP) pool. Gemcitabine can also become inactivated by dephosphorylation of the
monophosphate form by 5′-nucleotidases (5′-NTs), converting nucleotides back to
nucleosides. These enzymes play a critical role in the balance of dNTP pools and,
therefore, in gemcitabine metabolism. In this way, the rate-limiting step of
phosphorylation by dCK may be also be affected, compromising the overall beneficial
cytotoxicity of gemcitabine.
9
Figure 6. Gemcitabine cellular metabolism.18 hNT: human nucleoside transporter; dFdCMP: gemcitabine monophosphate; dFdCDP: gemcitabine diphosphate; dFdCTP: gemcitabine triphosphate; dFdU: 2′,2′-difluoro-2′-deoxyuridine, dFdUMP: 2′,2′-difluoro-2′-deoxyuridine monophosphate.
The most important mechanism of action of gemcitabine is inhibition of DNA
synthesis. When dFdCTP is incorporated into DNA, a single deoxynucleotide is
incorporated afterwards, and stops chain elongation. The non-terminal position of
gemcitabine makes DNA polymerases unable to proceed, as well as inhibits removal of
gemcitabine by exonucleases.
An additional mechanism of action of gemcitabine is inhibition of
deoxycytidylate deaminase (dCTD), by dFdCTP and indirectly by dFdCDP.23,24 By
covalently binding to the active site, dFdCDP inhibits Ribonucleotide reductase (RNR),
which catalyzes the reduction of ribonucleotides to deoxyribonucleotides.25-31 The
covalent inhibition of RNR causes decrease in the dNTP pool and consequently reduces
dCTD activity.32,33 In addition, decrease in the dNTP pool stimulates dFdC
phosphorylation, thereby increasing the level of dFdCTP, making dFdCTP more likely to
be incorporated into DNA competing with the NTPs.25,27 In addition, another important
mechanism of gemcitabine is the induction of apoptosis through caspase signaling. In
response to cellular stress, Gemcitabine activates p38 mitogen-activated protein kinase
(MAPK) to trigger apoptosis in tumour cells, but not in normal cells.
The widely accepted mechanism of covalent inhibition of RNR by dFCDP in the
presence and absence of reductant is shown in Figure-7.31 Crucial steps in RNR
mechanism include generation of tyrosine (Y122) radical from R2 subunit of RNR,
abstraction of a hydrogen radical from Cysteine (C439) in R1 subunit of RNR by Y122
ra
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11
preparation of numerous fluoro/gem-difluoro organic compounds.39,40 Various reagents or
reagent-combinations have been developed for oxidative desulfurization-fluorination
reactions over the years. Of which, desulfurization-difluorination of aldehyde and ketone
dithiolanes with oxidants such as N-halosuccinimides (NBS, NIS) or 1,3-dibromo-5,5-
dimethylhydantoin (DBH) and pyridinium poly(hydrogen fluoride) (PPHF)41 and
desulfurization-difluorination of alkyl aryl thioethers with DBH and Olah’s reagent
(Py.9HF)42,43 share similar mechanistic traits (Scheme 1).
Scheme 1. Oxidative desulfurization-difluorination of aldehyde, ketone dithiolanes; and alkyl aryl thioethers.
Hara’s research group has developed three reagents or reagent combinations for
oxidative desulfurization-fluorination reactions. First among them was Iodine
pentafluoride (IF5),44-47 which mainly causes the desulfurization-difluorination of benzyl
sulfides (Scheme 2). Later, an air- and moisture-stable IF5-pyridine-HF reagent48,49 was
developed for the desulfurization-difluorination of benzyl sulfides, thioacetals, and
dithianes. Recently, an air-stable fluorinating agent BrF3-KHF250 was also developed for
12
desulfurization-difluorination of benzyl sulfides, dithioacetals, and
(phenylthio)glycosides.
Scheme 2. Hara's oxidative desulfurization-difluorination of benzylic sulfides.
Interesting point to note is that none of the above mentioned methods provide
access to gem-difluoro products from alkyl aryl thioethers where in the thioether is
present on an internal secondary carbon atom (See Figure 13 in Section 2).
1.6. 5-Bromopyrimidines and 8-bromopurines: Syntheses and importance
The C-5 halogenated pyrimidine and C-8 halogenated purine nucleosides are
widely used as substrates in reactions involving direct displacement of halo groups with
nucleophiles.51,52 Especially, 5-bromopyrimidine and 8-bromopurine nucleoside
derivatives have been shown to possess interesting synthetic and biological
properties.51,52 Also, the halogenated pyrimidine and purine nucleoside derivatives are
often used in transition metal catalyzed cross-coupling reactions53 resulting in the
syntheses of a variety of nucleoside analogues with biological activity and/or fluorescent
probes.54 For example, a number of 5-substituted uracil derivatives, especially
arabinofuranosyl- and 2'-deoxyuridines, have been investigated extensively for the
clinical treatment of viral diseases.55 The 5-bromo and 5-iodo uridine derivatives are used
as substrates for high yield coupling with terminal alkynes to generate 5-alkynyluracil
nucleosides with antiviral activity56,57 and the 5-alkynyl products can also be transformed
into bifurane derivatives which possess potent and selective inhibition of Varicella-Zoster
13
virus.56,58 Moreover, radiolabeled 5-bromo- and 5-iodouracil nucleosides have
applications in cellular biochemistry.59
Halogenated pyrimidine52 and purine51 nucleosides have been prepared by direct
treatment with halogens or halonium ion (X+) sources but most of these methods utilize
harsh reaction conditions. Various C5-bromouridine derivatives have been synthesized
using Br2/Ac2O/AcOH,52 Br2/H2O,60 N-bromosuccinimide (NBS) in DMF61 or ionic
liquids,62 combination of 3-chloroperoxybenzoic acid/HBr in aprotic solvents,63 ceric
ammonium nitrate (CAN)/LiBr in protic or aprotic solvents,64 or KBr/Oxone.65 The C5-
bromination of cytidine nucleosides has been accomplished with Br2/CCl4/hν66 or NBS in
DMF61 or ionic liquids.62 On the other hand, the synthesis of C8-bromo purine
nucleosides has been typically achieved with Br2/AcOH/AcONa67 or NBS/DMF.61
Hence, a universal brominating reagent for C5-bromination of pyrimidines and C8-
bromination of purines and also development of a bench-friendly bromination
methodology is warranted.
1.6.1. 1,3-dibromo-5,5-dimethylhydantoin, an important brominating reagent
The 1,3-dibromo-5,5-dimethylhydantoin (DBDMH or DBH, Figure 8) is a useful
reagent for various organic transformations68-70 including aromatic bromination.71-75
Enhanced efficiency of DBH towards aromatic bromination in the presence of acids has
been known.72-74 Moreover, Lewis acid-catalyzed benzylic bromination with DBH has
also been reported.76 DBH also efficiently oxidizes thiols to disulfides with DBH.77,78 In
addition, α-bromination of aliphatic ketones is achieved with DBH in combination with
p-toluenesulfonic acid (TsOH).79 Interestingly, compared to NBS, DBH can donate two
bromines and can be doubly efficient.
14
Figure 8. Structures of DBH and NBS
1.7. Selected aspects of the chemistry of 7-deazapurine nucleosides
The 7-deazapurine nucleosides, namely, tubercidin, sangivamycin and
toyocamycin (19a-c, Figure 9) are a group of natural products isolated from the species
Streptomyces. Analogues and derivatives of these 7-deazapurine nucleoside antibiotics
have been synthesized and subjected to extensive biological testing.80-84 Chemical
modifications of the parent 7-deazapurine antibiotics as well as sugar-base coupling has
provided access to a sizeable number of biologically active 7-deazapurine compounds.85-
96
Figure 9. Structures of 7-deazapurine nucleosides
Noteworthy examples of such molecules include: (a) 5-iodotubercidin 20,97 an up-
field activator of the p53 pathway; (b) sangivamycin analogues such as 6-
hydrazinosangivamycin 2298 and 4-Amino-6-bromo-7-(β-L-xylofuranosyl)pyrrolo[2,3-
d]pyrimidine-5-carboxamide (xylocidine, 23),99 in vitro down-field inhibitors of PKC and
CDK in cancer cell lines; (c) a methyl-substituted tubercidin 21,100 which acts against the
15
replication of polio and dengue viruses; (d) the anti-HSV agent xylotubercidin;101 (e)
substituted toyocamycin analogues 24102 and 2'-β-C-methyl derivative of toyocamycin,103
sangivamycin,103 and tubercidin104,105 that have activity against HCV; (f) 2'-deoxy-2'-
fluoroarabinotubercidin106 and 2-amino-2'-deoxy-2'-fluoroarabinotubercidin,107,108 which
exhibit antiviral activity; (g) 4N,5-diaryltubercidin derivatives 25, which act as adenosine
kinase inhibitors;96,109,110 and (h) tubercidin derivatives such as 4-(het)aryl,111 5-
(het)aryl112 and some 4-substituted-5-(het)aryl113 compounds 26 with nanomolar
cytostatic activity against several cancer cell lines. Important base-modified analogues
are shown in Figure 10.
Figure 10. Important base-modified 7-deazapurine nucleoside derivatives.
Direct alkylation of exocyclic amino groups on 7-deazapurine nucleosides has not
been noted. However, 4-N-methyltubercidin (6-N-methyl-7-deazaadenosine) 28 was
reported to be synthesized from the reaction of tubercidin with methyl iodide followed by
1N sodium hydroxide (Scheme 3).86 Thus, treatment of tubercidin with CH3I (rt, 24 h,
16
DMA) gave the corresponding N1-methyltubercidin hydroiodide 27 after recrystallization
from methanol. Base treatment of this cationic intermediate (1N NaOH 100 oC, 1.25 h)
produced 4-N-methyltubercidin 28 via Dimroth rearrangement.86
Scheme 3. Synthesis of 4-N-methyltubercidin by Dimroth-rearrangement approach
Even though direct alkylation or Dimroth type rearrangement has been scarce in
7-deazapurine chemistry, the 4-N-substituted-7-deazapurine derivatives have been widely
prepared by aromatic nucleophilic substitution (SNAr). For example, SNAr displacement
of chloro group from 4-cholo analogues86,113 or triazolyl group from 4-N-(1,2,4-triazol-4-
yl) intermediates114 gave access to various other 4-N-modified tubercidin derivatives.
Also, 4-N-substituted toyocamycin derivatives were prepared from the SNAr
displacement of chloro group from the corresponding 4-chloro compounds.102 Likewise,
the 7-N-benzylformycin was prepared via 7-chloro derivative of the C-nucleoside
antibiotic formycin (3-β-D-ribofuranosylpyrazolo[4,3-d]pyrimidine).115
1.8. Nucleoside transport inhibitors
As discussed in previous sections (Chapter 1, Sections 1.1-1.3), nucleoside based
drugs have been in use for the treatment of various infections. Since the hydrophilic
nature of nucleosides limits their cell permeability, nucleoside-specific membrane
transport carriers (NT) facilitate and regulate the cellular uptake and therapeutic actions
17
of many nucleoside based drugs.116,117 The nucleoside transporter proteins are classified
into two categories: equilibrative NT (ENT) and concentrative NT (CNT). Human
equilibrative NTs (hENTs) are found in the outer plasma membrane and some
intracellular membranes of most, if not all, human cells.118
Nitrobenzylmercaptopurine ribonucleoside (NBMPR, 29a) and structurally
related hENT1 probes such as 6-N-(4-nitrobenzyl)adenosine (29b)119 and 5'-S-{2-(6-[3-
(fluorescein-5-yl)thioureido-1-yl]hexanamido)}ethyl-6-N-(4-nitrobenzyl)-5'-
thioadenosine (FITC-SAHENTA)120 inhibit hENT1 with nM affinity (Figure 11).
Discovery and development of such nucleoside transport inhibitors has greatly aided
investigations on the structure, function, and cell surface abundances of hENT1
proteins.116,120,121
Figure 11. Structures of Important nucleoside transport inhibitors.
2. RESEARCH OBJECTIVES
(i) The first research objective of this dissertation was to develop novel fluorination
methodologies for the synthesis of mono-fluoro and geminal-difluoro uridine analogues
from their parent nucleoside precursors. These strategies were envisioned to eliminate the
current need to couple fluorinated sugar precursors with nucleosides bases, a process
which has severe regiochemical and stereochemical limitations. On the basis of literature
18
precedence,122 I would explore the possibility of insertion of the fluorine or geminal-
difluoro unit into the 2'-position of ribose moiety of nucleosides using oxidative
desulfurization-fluorination of substrates of type 30 and 31 with 1,3-dibromo-5,5-
dimethylhydantoin (DBDMH or DBH) and Olah’s reagent (Py.9HF) (Figure 12).
Figure 12. Different classes of 2'-arylthionucleosides for oxidative desulfurization-fluorination and reductive desulfonylation-fluorination procedures.
A complementary approach was designed based on reductive-
desulfonylation/fluorination processes of 2'-arylsulfonyl-2'-deoxyuridine 32 or 2'-
arylsulfonyl-2'-deoxy-2'-fluorouridine 33 with electrophilic fluorine reagents (Figure 12).
Treatment of sulfone 32 or α-fluorosulfone 33 with organic electron donors such as
tetrakis(dimethylamino)ethylene (TDAE), Murphy’s reagent, would be expected to lead
to the reductive cleavage of sulfone moiety and generation of C2'-centered carbanion.
The intermediate carbanion would then be quenched with fluoronium ion (F+) to give
mono- or di- fluorinated uridine derivatives. Alternatively, treatment of 2'-arylsulfonyl-2'-
deoxyuridine 32 with a base would lead to abstraction of the most acidic proton H2' and
then the intermediary carbanion would be quenched with electrophilic fluorine (F+) to
give products of type 33.
19
(ii) Since nucleosides are complex molecules and also because of the lack of literature
precedence for the desulfurization-difluorination of aryl-alkyl thioethers wherein the
arylthio unit is positioned on a secondary internal carbon atom, I planned to perform
model studies on simpler aryl-alkyl thioether substrates of type 34-37 (Figure 13). The
proposed substrates would have different functional groups such as ester (34), keto (35),
alkane (36) and lactone (37), and mimic the C2' of ribose unit in the pyrimidine
nucleoside precursor (30, Figure 12), which also has a carbon at the carbonyl oxidation
state (as an aminal group) on the α-carbon atom.
Figure 13. Various aryl-alkyl thioether substrates for model oxidative desulfurization-difluorination reactions
(iii) Since DBH is a source of electrophilic bromine (Br+) and as bromonium ions are
known to brominate pyrimidines at C-5 position and purines C-8 position, I explored the
bromination at C-5 of pyrimidines and C-8 of purines with DBH. The goal of this part of
my dissertation was to develop an efficient bromination methodology, which could be
applicable for the bromination of all DNA and RNA nucleosides, and which would also
be compatible with commonly used protection groups in nucleoside chemistry.
(iv) The next goal of my dissertation was to investigate the behavior of gemcitabine and
its 2'-modified analogues such as 2'-deoxy-2'-fluorocytidine (38) and 2′-deoxy-2′-fluoro-
2′-C-methylcytidine (PSI-6130, 39) in one electron oxidation systems and evaluate if
20
stereo electronic differences at C2'-position among these substrates would allow us to
detect C2' and/or C3' sugar radicals using electron spin resonance (ESR) spectroscopy
studies (Figure 14). This part of my dissertation was planned in collaboration with Dr.
Michael D. Sevilla’s research group of Oakland University.
Figure 14. Gemcitabine and its analogues for one electron oxidation studies
(v) Since 6-N-(4-nitrobenzyl) derivatives of adenosine and other adenine nucleosides
possess potent nucleoside transport inhibition activity, in the next goal, I proposed to
synthesize novel 6-N-benzyl-7-deazapurine nucleoside derivatives (40a-d) of 7-
deazaadenosine antibiotics (19a-c) in order to test their biological activity (Figure 15). I
envisioned two methods for achieving the synthesis of the target molecules (40a-d). One
involved alkylation of the 7-deazaadenosine antibiotics (19a-c) at N1 with a benzyl
bromide followed by a base-promoted Dimroth rearrangement. The second route
employed diazotization–fluorodediazoniation followed by SNAr displacement of fluoride
with a benzyl amine.
21
Figure 15. Structures of 7-deazapurine antibiotics and the proposed 6-N-benzyl
analogues.
(vi) I also intended to synthesize 7-deazapurine nucleoside derivatives with various novel
modifications at C-8 and C-7 position (Figure 16). For example, 8-bromo-7-deazapurine
derivatives (41, Y = Br) are envisioned as substrates for the synthesis of novel 8-azido-7-
deazapurine analogues (41, Y = N3). These 8-azido-7-deazapurine nucleosides might act
as substrates for Copper-catalyzed and copper-free click reactions with alkynes and
cycloalkynes respectively. The resultant novel 8-triazolyl-7-deazapurine derivatives (41,
Y = triazolyl) might possess fluorescent properties because of extended conjugation.
Figure 16. Structures of various 8-modified-7-deazapurine derivatives
Also, the 8-bromo-7-deazapurine substrates (41, Y = Br) can be converted to the
corresponding 8-alkynyl-7-deazapurine counterpart 42. The crucial 8-alkynyl
intermediates 42 might give access to α-halovinyl (43, Z = Cl, Br), β-ketosulfone (43, Z =
22
OH), and α-vinylazide (44) derivatives, analogous to recent findings on similar
modifications at C5-position of pyrimidines (unpublished results from our research
group).
3. RESULTS AND DISCUSSION
3.1. Bromination of nucleobases with 1,3-dibromo-5,5-dimethylhydantoin
Since DBH is known to be a good source of bromonium ion (Br+), I envisioned a
novel bromination protocol toward C5-bromination of pyrimidines and C8-bromination
of purines.
3.1.1 Bromination at C5 position of pyrimidine nucleosides with DBH
To test my hypothesis, I initially treated 2',3',5'-tri-O-acetyluridine 45a with 1.1
equivalents of DBH in CH2Cl2 at ambient temperature (Scheme 4; Table 3, entry 1).
After 28 h of reaction time TLC showed conversion of most of the substrate to a new
spot, which was less polar than the substrate spot. The less polar spot was isolated by
aqueous workup (95%) and 1H-NMR of the crude material was very clean and implied a
single compound. Characteristic doublet for H5 at 5.73 ppm was missing in 1H-NMR and
the doublet for H6 (7.33 ppm) collapsed to a singlet (7.82 ppm), indicating the formation
of 2',3',5'-tri-O-acetyl-5-bromouridine (46a; 95%). Furthermore, structure of the product
was confirmed by comparing NMR data with literature NMR data for 2',3',5'-tri-O-
acetyl-5-bromouridine.64 To optimize the amount of DBH required for the bromination, I
treated 45a with 0.55 eqiuv. of DBH. But the TLC showed only 60% conversion to 46a
even after 48 h of reaction time. Although, two bromonium ions (Br+) can be delivered
by one equivalent of DBH, it seemed that 1.1 equiv. of DBH is required for complete
conversion of 45a to 46a at room temperature.
23
Role of Lewis acids
Since it was reported that Lewis acids enhance the efficiency of bromination by
DBH,76 I decided to compare the rate of bromination in presence of Lewis acids.
Remarkably, treatment of 45a with 0.55 equiv. of DBH in presence of 0.55 equiv. of
trimethylsilyl trifluoromethanesulfonate (TMSOTf) significantly enhanced the rate of
bromination yielding 46a after only 6 h (94% yield) (entry 2). The efficiency of
bromination did not differ much when 1.1 equiv. of TMSOTf was used (entry 3). Also,
replacing TMSOTf with other organic acids such as p-toluenesulfonic acid (TsOH, 0.75
equiv.) required 0.75 equiv. of DBH for complete conversion of 45a to 46a (entry 4).
Next, I studied the effect of temperature and role of solvents on bromination in order to
establish optimal reaction conditions.
Scheme 4. Bromination of uracil-derived nucleosides 1 with 1,3-dibromo-5,5-dimethylhydantoin (DBH). See Table 3 and 4 for specific reaction parameters.
Effect of Temperature
Treatment of 45a with equimolar quantities of DBH and TMSOTf (0.55 equiv.) at
40 oC in CH2Cl2 afforded 46a quantitatively in only 2 h (entry 5). Whereas, analogous
24
treatment of 45a with DBH/TMSOTf at 0 oC gave 46a in only 65% yield after 8 h (TLC)
and required 1.1 equiv. of DBH for complete conversion (entry 6).
Role of Solvents
Treatment of 45a with 0.55 equiv. of DBH at room temperature in CH3CN gave
46a after 11 h (entry 7). Analogous treatment of 45a with DBH in presence of TMSOTf
(0.55 equiv.) yielded 46a after only 2.5 h (entry 8). Moreover, the DBH/DMF system was
much more effective than CH3CN or CH2Cl2 affording 46a in much shorter time (entries
9-10). In addition, bromination of 45a with DBH in the presence or absence of TMSOTf
was less efficient in polar protic solvent MeOH, producing 46a in low yields. Hence, it
can be concluded that bromination of 45a with DBH and/or TMSOTf was very efficient
in polar aprotic solvents such as CH3CN or DMF. However, it is noteworthy that prior
removal of these polar solvents from crude reaction mixture was required before
proceeding to aqueous workup, which is not the case with CH2Cl2.
Table 3. Effect of various reaction parameters on C5-bromination of 2',3',5'-tri-O-
acetyluridine 45a with DBHa
Entry Solvent Temp.
(oC)
DBH
(equiv.)
TMSOTf
(equiv.)
Time
(h)
Yieldb,c
46a (%)
1 CH2Cl2 25 1.1 - 28 95
2 CH2Cl2 25 0.55 0.55d 6 94
3 CH2Cl2 25 0.55 1.10 6 91
4 CH2Cl2 25 0.75 0.75e 8 94
5 CH2Cl2 40 0.55 0.55 2 98
25
6 CH2Cl2 0 1.1f 0.55 3 98
7 CH3CN 25 0.55 - 11 86g
8 CH3CN 25 0.55 0.55 2.5 90
9 DMF 25 0.55 - 0.6 95
10 DMF 25 0.55 0.55 0.3 98
a Bromination was performed on 0.1 mmol scale of 45a. b Isolated yield after aqueous work-up. c Purity of the product 46a was determined by TLC and 1H NMR and was higher than 97% unless otherwise noted. d Reaction without TMSOTf showed 60% conversion to 46a (TLC) after 48 h and complete conversion after 68 h with purity over 90% (1H NMR). e TsOH was used instead of TMSOTf. f Reaction with 0.55 eq. of DBH was complete in 65% after 8 h. g With purity over 90%.
Extension to other protected uridine nucleosides
I also wanted to test the applicability of the optimized bromination procedure for
the 5-bromination of the other uracil nucleosides. Thus, treatment of 1-(2,3,5-tri-O-
acetyl-β,D-arabinofuranosyl)uracil 45b with DBH and TMSOTf yielded the
corresponding C5-brominated analogue 46b in 91% yield after 10 h (Scheme 4; Table 4,
entry 2). Replacing CH2Cl2 with polar aprotic solvent such as CH3CN generated 46b in
98% yield after only 2 h (entry 3). In addition, treatment of 3',5'-di-O-acetyl-2'-
deoxyuridine 45c with DBH produced the 5-bromo counterpart 46c in 72% isolated yield
after 18 h (entry 4). 46c was also obtained after only 2.5 h in the presence of TMSOTf
(entry 5). Moreover, the rate of bromination increased five-fold when the reaction was
carried out at 40 oC (entry 6).
C5-Bromination of unprotected uridine nucleosides with DBH
26
On the basis of the successful C5-bromination of protected uridine nucleosides, I
envisioned the extension of DBH bromination protocol for the C5-bromination of
unprotected uridine nucleosides. Thus, bromination of uridine 45d with DBH in DMF
yielded 5-bromouridine 46d in 75% crystallized yield after only 20 minutes (Scheme 4;
Table 4, entry 7). Analogously, treatment of 1-(β,D-arabinofuranosyl)uracil 45e and the
acid sensitive 2'-deoxyuridine 45f with DBH at ambient temperature produced the
corresponding 5-brominated products in good yields (entries 8 and 9).
Extension to cytidine nucleosides
With the successful application of C5-bromination of uridine nucleosides with
DBH, I imagined the extension of the DBH-bromination methodology toward the
synthesis of C5-brominated cytidine analogues. Thus, treatment of cytidine 47a with
0.55 equiv. of DBH at room temperature in DMF produced 5-bromocytidine 47b after
only 30 minutes (72% yield, Figure 17; Table 4, entry 10).
Table 4. Bromination at C5 position of the pyrimidine based nucleosides 45a-f and 57-59a
Entry Substrate Product SolventTemp.
(oC)
DBH
(equiv.)
TMSOTf
(equiv.)
Time
(h)
Yieldb
(%)
1 45a 46a CH2Cl2 25 0.55 0.55 6 94c
2 45b 46b CH2Cl2 25 0.55 0.55 10 91c
3 45b 46b CH3CN 25 0.55 0.55 2 98c
4 45c 46c CH2Cl2 25 1.10 - 18 72d
5 45c 46c CH2Cl2 25 0.55 0.55 2.5 90c
27
a Bromination was performed on 0.25-2.0 mmol scale. b Isolated yield. c After aqueous work-up with purity higher than 97% (1H NMR). d After column chromatography. e After crystallization. f Direct crystallization of the crude reaction mixture from MeOH gave 48b in 46% yield.
Analogously, bromination of 4-N-benzoylcytidine 48a proceeded smoothly
generating 48b (entry 11), which implies that the presence of an electron withdrawing
group on the cytosine ring (N4-benzoyl) does not affect the efficiency of bromination.
Protection group compatibility
I also found that DBH bromination methodology was compatible with common
protection groups used in nucleoside chemistry. Thus, treatment of 5'-O-(tert-
butyldimethylsilyl)-2',3'-O-isopropylideneuridine 49a with 0.55 equiv. of DBH in
DMF afforded the corresponding 5-bromo product 49b in quantitative yield (Figure
17; Table 4, entry 12). It was already shown that base-labile acetyl groups were stable
during DBH-bromination (e.g., Table 3). Therefore, it can be concluded that acid
6 45c 46c CH2Cl2 40 0.55 0.55 0.5 93c
7 45d 46d DMF 25 0.55 - 0.33 75e
8 45e 46e DMF 25 0.55 - 1 65d
9 45f 46f DMF 25 0.55 - 0.75 80d
10 47a 47b DMF 25 0.55 - 0.5 72d
11 48a 48b DMF 25 0.55 - 0.5 74d, f
12 49a 49b DMF 25 0.55 - 0.5 98c
13 45d 46d MeOH 25 0.55 - 1 70e
14 47a 47b MeOH 25 0.55 - 1 68d
28
labile as well as base labile protection groups are stable toward DBH-bromination
protocol.
Figure 17. Selected pyrimidine nucleoside precursors (series a) and their brominated products (series b).
C5-Bromination of unprotected pyrimidine nucleosides with DBH in MeOH
Since DMF has to be removed from the crude reaction mixture before purification
of the brominated products, I used a low-boiling polar solvent such as MeOH as a
replacement for DMF. To my surprise, treatment of uridine 45d with DBH (0.55 equiv.)
in MeOH at room temperature for 1 h yielded 5-bromouridine 46d in 70% yield
(crystallization) (Scheme 4; Table 4, entry 13). Analogously, cytidine 47a was also
brominated using DBH in MeOH generating 5-bromocytidine 47b (Figure 17; Table 4,
entry 14) after only 1 h in 68% yield (column chromatography). Therefore, it can be
concluded that DBH/MeOH system offers a convenient access to the unprotected 5-
bromopyrimidine nucleosides.
3.1.2 Bromination at C8 position of purine nucleosides with DBH
Next, I planned the extension of the DBH and DBH/TMSOTf combination toward
the synthesis of 8-bromo purine nucleosides. Thus, treatment of adenosine 50a with DBH
(1.75 equiv.) in DMF at room temperature gave 8-bromoadenosine 50b (48% yield) after
5 h at ambient temperature (Figure 18, Table 5, entry 1). Analogous treatment of 2'-
29
deoxyadenosine 51a with DBH (1.5 equiv.) afforded 8-bromo-2'-deoxyadenosine 51b in
68% yield in 3.5 h (entry 2).
Figure 18. Selected purine nucleoside precursors (series a) and their brominated products (series b).
The 2',3',5'-tri-O-acetylguanosine 52a was converted to the 8-bromo derivative
52b upon treatment with 0.55 equiv. of DBH in DMF at room temperature for 2.5 h
(entry 3). Replacement of DMF with CH3CN decreased the rate of bromination yielding
52b after 4 h at room temperature (entry 4). On the other hand, treatment of guanosine
with 0.75 equiv. of DBH in DMF at ambient temperature produced 8-bromoguanosine in
48% yield after recrystallization from water (entry 5). Use of TMSOTf did not improve
the yield of 8-bromoguanosine 53b, though less equiv. of DBH was required for
complete conversion (entry 6). Treatment of inosine or acetyl protected inosine with
DBH or DBH/TMSOTf in DMF failed to afford 8-bromoinosine, which was in
accordance with the reported failed bromination attempts of inosine with NBS in
DMF.123
30
Table 5. Bromination of selected purine nucleosides at ambient temperature (see Figure 11 for structures)a
aBromination was performed on 0.5-1 mmol scale. b Isolated yield. c After column chromatography. d Reaction showed formation of the product in approximately 80% yield (TLC). e Isolated yield after aqueous work-up. f Reaction with 0.55 equiv. of DBH was completed in 24 h. g After crystallization from water. Bromination was quantitative as judged by TLC. i After filtration from reaction. j Reaction showed formation of the product in approximately 70% yield (TLC).
In conclusion, DBH and/or DBH/TMSOTf also effected bromination of purine
nucleosides at the C8-position, although reactions usually required higher equivalency of
DBH, longer reaction times, and produced the corresponding brominated products in
lower yields when compared to the pyrimidine analogues.
C8-Bromination of unprotected purine nucleosides with DBH in MeOH
Entry Substrate Product SolventDBH
(equiv.)
TMSOTf
(equiv.)
Time
(h)
Yieldb
(%)
1 50a 50b DMF 1.75 - 5 48c,d
2 51a 51b DMF 1.50 - 3.5 68c,d
3 52a 52b DMF 0.55 - 2.5 83c
4 52a 52b CH3CN 0.55 - 4 98e
5 53a 53b DMF 0.75f - 2.5 51g
6 53a 53b DMF 0.60 0.55 0.5 48g
7 50a 50b MeOH 1.75 - 16 47c, j
8 51a 51b MeOH 1.50 - 3.5 70j
9 53a 53b MeOH 0.55 - 1 50g
10 54a 54b MeOH 0.55 - 0.5 72i
31
Next, I designed the extension of the DBH/MeOH system toward the synthesis
of 8-bromo purine nucleosides. Thus, treatment of adenosine 50a with DBH (1.75
equiv.) in MeOH at room temperature gave 8-bromoadenosine 50b (47% yield) after
16 h at ambient temperature (Figure 18, Table 5, entry 7). Analogous treatment of 2'-
deoxyadenosine 51a with DBH (1.5 equiv.) showed only ~70% conversion to 8-
bromo-2'-deoxyadenosine 51b (entry 8, TLC scale reaction). Interestingly, treatment
of guanosine 53a with DBH (0.55 equiv.) in MeOH at room temperature yielded 8-
bromoguanosine 53b in 50% yield after recrystallization of the crude reaction mixture
from water (entry 9). In contrast, treatment of 2'-deoxyguanosine 54a with DBH in
MeOH at room temperature for 30 minutes led to the solidification of product 54b
from the reaction. The resultant solid was collected by vacuum filtration and was
>95% pure on 1H-NMR (entry 10). In conclusion, DBH/MeOH system works very
efficiently with guanine nucleosides and moderately efficient with adenine
nucleosides.
3.1.3. Bromination at C8 position of 7-deazapurine nucleosides with NBS and DBH
Encouraged by my findings of bromination of purine nucleosides at C8 position
with DBH, I further explored possibility of bromination of 7-deazapurine antibiotics
(19b-c) with DBH as well as with NBS (Scheme 5). Previously, 8-bromotoyocamycin
(55) was prepared either from coupling 8-bromo-7-deazapurine derivative to the ribose
sugar derivative124 or by treating 19c with Br2/H2O125. I achieved the synthesis of
compound 55 by two procedures, both proceeding by electrophilic aromatic substitution.
One involved electrophilic aromatic substitution using N-bromosuccinimide (NBS) in
anhydrous dimethylformaldehyde (DMF). The second route employed substitution using
32
1,3-dibromo-5,5-dimethylhydantoin (DBH/DBDMH) in MeOH. Thus, treatment of 19c
with NBS in DMF at ambient temperature for 2.5 h yielded 55 in 45% after column
chromatography. Reaction took 4 h to complete when DMF was replaced with MeOH to
give 55 in 64% after recrystallization. However, bromination of 19c using DBH in
MeOH proceeded efficiently in 30 min by which time product solidified out of solution,
was collected by vacuum filtration and the mother liquor was recrystallized to give 55 in
85% overall yield. The DBH/MeOH method was the most efficient of the three for
synthesizing 55 in terms of yield, reaction time, product isolation.
Scheme 5. Synthesis of 8-bromo-7-deazanucleosides. Reagents and conditions: d) NBS (1.1-1.5 eq), DMF, rt, 1-2.5 h; e) DBH (0.75-1.1 eq), MeOH, rt, 30-45 min.
Over the years, the synthesis of 8-bromosangivamycin 56 has mainly been
achieved by conversion of 55 to 56 using concentrated ammonium hydroxide and 50%
hydrogen peroxide solution.124 Owing to the successful preparation of 55, we envisioned
the synthesis of 56 using NBS/DMF and DBH/MeOH systems. Thus, treatment of
sangivamycin (19b) with NBS in DMF at ambient temperature for 1.5 h gave 8-
bromosangivamycin (56) in 63% yield after column chromatography. Even though the
reaction proceeded faster in DBH/MeOH system (30 min) the yield was low (40%),
which can be attributed to the presence of some unidentified byproducts. Fortunately,
there is literature precedence for convenient synthesis of 56 from 55.124 Thus, treatment
33
of 55 with NH4OH/50%H2O2 system gave 56 in 86% yield after vacuum filtration from
the reaction mixture. In summary, an efficient bromination methodology for the
synthesis of 8-bromotoyocamycin and 8-bromosangivamycin was developed using
NBS/DMF and DBH/MeOH systems.
3.1.4. Plausible mechanism of bromination of nucleobases with DBH
Bromination of nucleosides using DBH is expected to proceed via aromatic
electrophilic bromination pathway with bromonium ion (Br+) as the bromine source from
DBH.
Plausible mechanism for C-5 bromination of pyrimidines by DBH
Carbocation intermediate (type A) is generated upon reaction of pyrimidine base
57 with DBH (Scheme 6). The intermediate A is highly unlikely to exist in resonance
with the intermediate B, because of the presence of electropositive carbonyl group
adjacent to the N1 centered cation. Abstraction of the proton from C5 by the anion C
regenerates the aromaticity, thus providing the 5-bromopyrimidine product 58.
R
N
HN
O
O N
NO
Br
BrO
R
N
HN
O
O
Br
H
H N
NO
BrO
R
N
HN
O
O
A
Br
C
R
N
HN
O
O
Br
H
H
B
57 DBH 58
Scheme 6. Plausible mechanism of C-5 bromination of pyrimidine nucleosides by DBH.
Plausible mechanism for C-8 bromination of purines by DBH
34
I hypothesize that the N7-C8 double bond of purine 59 abstracts a bromonium ion
(Br+) from DBH to give nitrogen centered cation A, which is stabilized by three
resonance structures B-D (Scheme 7). Then aromaticity is regenerated by abstraction of
proton at C8 position in A', probably by hydantoin intermediate E, to give the 8-
bromopurine product 60.
Scheme 7. Plausible mechanism of C-8 bromination of purine nucleosides by DBH.
Plausible mechanism for TMSOTf activation of DBH:126
Upon reaction with TMSOTf (61), DBH generates a silylenol intermediate A
along with a reactive bromonium triflate (TfOBr) intermediate B. Intermediate B acts as
the actual bromonium ion (Br+) source for the bromination of the pyrimidine/purine
substrate 62 to generate the corresponding brominated product 63 along with triflic acid
(TfOH, C) (Scheme 8). Triflic acid C is reacts with the silylenol intermediate A to give
another silylenol intermediate D and one more equivalent of TfOBr (E). Second
equivalent of substrate 62 gets brominated with E to release TfOH (F), which then reacts
with the silylenol intermediate D to give 5,5-dimethylhydantoin (DMH, G) and TMSOTf.
35
Scheme 8. Plausible mechanism of TMSOTf enhanced DBH bromination.
3.2. Attempted synthesis of 2'-deoxy-2',2''-difluoropyrimidine nucleosides
Because of the ease of preparation of 2'-S-aryl-2'-deoxy-2'-thiouridine (64, Figure
19) and 2'-deoxy-2'-α-fluorosulfone derivative (65), I explored the possibility of
introduction of mono and/or difluoro unit into the C2' position using oxidative
desulfurization-(di)fluorination and reductive desulfonylation-fluorination methods. The
oxidative fluorination processes were envisioned to proceed via (i) desulfurization-
difluorination of the uridine-2'-thioethers (substrate 64, X = H) or (ii) desulfurization-
fluorination of uridine α-fluorothioethers (substrate 64, X = F) by using halonium ion
(Br+, I+) reagents as oxidants and a nucleophilic fluorine source (F-) for quenching the
intermediary C2'-carbocation. Whereas, the reductive fluorination processes were
proposed to proceed via (i) abstraction of the C2'-proton (from substrate 65, X = H) using
a base and quenching the intermediary C2'-carbanion with electrophiles such as F+, H+,
CH3+, etc.; or (ii) cleavage of the sulfonyl moiety (from substrate 65, X = H or F) with
single electron transfer reagents and quenching the resultant C2'-carbanion with
electrophiles such as F+, H+, CH3+, etc.
36
O
RO
RO
N
N
O
O
S Z
R = Ac, R' = H or R, R' = Bn; Z = Cl, OCH3
R'
O
RO
RO
N
N
O
O
S
R'
X
ZO
O
X
X = H or F
O
RO
RO
N
N
O
O
F
R'
XOxidative
desulfurization-(di)fluorination
Reductive
desulfonylation-fluorination
64 66 X = H, F65
X = H, F
1'
2'2'
1'
Figure 19. General scheme for the proposed C2'-quarternization of 2'-arylthiouridine derivatives
3.2.1. Oxidative desulfurization-fluorination approaches
The oxidative fluorination approach is modeled on Haufe’s desulfurization-
difluorination of aryl-alkyl thioethers using DBH and Olah’s reagent (Py.9HF) for the
synthesis of gem-difluoroalkanes (Scheme 9).42,43
Scheme 9. General scheme for Haufe’s desulfurization-difluorination of aryl-alkyl thioethers.
3.2.1.1. Model desulfurization-fluorination studies
To apply Haufe’s desulfurization-difluorination methodology to nucleoside
substrates, it is necessary to test the general applicability of the method on model
compounds first. As the C2'-position, where the thioether functionality is present, is in α-
position to an aminal carbon, substrates with an aminal or electron withdrawing group on
the α-position should be used in model studies. Another reason for conducting model
reactions on such substrates which would mimic C2' of ribose unit in the pyrimidine
nucleoside substrates is because all previous desulfurization-difluorination protocols were
successful only for the compounds where the sulfenyl/phenylthio group is present on the
37
terminal and primary carbon. Therefore, I developed a desulfurization-difluorination
protocol of α-thioesters, where in the sulfenyl group is present in the middle of the chain
on a secondary carbon.
Initially, open chain aryl-alkyl thioethers (of type 72-75, Scheme 10) with an ester
group on the α-position (prepared conveniently from commercially available ethyl 2-
bromoalkanoate 71a or 71b) were tested for desulfurization-difluorination protocol.
Thus, treatment of ethyl 2-(phenylthio)octanoate 72 with DBH (3 eq.) and Olah’s reagent
(6 eq.) in CH2Cl2 at ambient temperature for 2 h gave ethyl 2,2-difluorooctanoate 77 in
80% yield after only aqueous workup (Table 6, entry 1). However, analogous treatment
of 72 with DBH/Py.9HF for 4 h gave 77 in 90% yield after aqueous workup (entry 2). To
study the effect of temperature, phenylthioether 72 was treated with DBH (3 eq.) and
Olah’s reagent (6 eq.) in CH2Cl2 at 35 oC and the difluoro product 77 was obtained in
85% yield after only 1 h (entry 3). But 77 was obtained in 95% yield after stirring the
reaction for 2 h at 35 oC (entry 4).
Scheme 10. Desulfurization-difluorination of α-thioesters.
To test the influence of the substituent on the para position of aromatic ring of the
thioether substrates, 4-chlorophenyl thioether (73-74) and 4-methoxyphenyl thioether
(75) were selected. Thus, treatment of ethyl 2-((4-chlorophenyl)thio)hexanoate 73 with
38
DBH (3 eq.) and Olah’s reagent (6 eq.) in CH2Cl2 at ambient temperature for 1 h gave
ethyl 2,2-difluorohexanoate 76 in 80% yield after only aqueous workup (Table 6, entry
5). However, difluoro ester 76 was obtained in 90% yield when the reaction was stirred
for 2 h (entry 6). In addition, treatment of 4-chlorophenyl thioether 73 with DBH (3 eq.)
and Olah’s reagent (6 eq.) in CH2Cl2 at 35 oC generated the difluoro product 76 in 95%
yield after only 1 h (entry 7). Interestingly, no geminal bromo-fluoro by product was
observed in crude NMR and GC-MS. When less equivalents of DBH (3 equiv. vs 2
equiv.) and Olah’s reagent (6 equiv. vs 4 equiv.) were used, decreased yield of the
difluoro product 76 (80%) was observed (entry 8). Thus, 3 equiv. of DBH and 6 equiv. of
Olah’s reagent were found to be optimal for the desulfurization-difluorination reactions.
DBH vs NBS vs NIS
Treatment of ethyl 2-((4-chlorophenyl)thio)octanoate 74 with N-
bromosuccinimide (NBS; 3 eq.) and Olah’s reagent (6 eq.) in CH2Cl2 at 35 oC for 2 h
followed by aqueous workup showed complex 1H-NMR spectrum and the
difluoroocatnoate 77 was only found to be 25% according to the 19F-NMR of the crude.
There was no increase in % of difluoro product 77 even after stirring for 4 h at 35 oC
(entry 9). Analogous treatment of 74 with N-iodosuccinimide (NIS; 3 eq.) and Olah’s
reagent (6 eq.) in CH2Cl2 at 35 oC for 24 h gave very complex 1H-NMR and 19F-NMR,
with the difluoroproduct 77 was found to be 5% (entry 10). Thus, DBH was proven to be
more effective than other halogen oxidants such as NBS or NIS, as observed by Haufe, et
al.43
Table 6. Desulfurization-difluorination studies of α-thioester derivatives with DBH/Py.9HFa
39
Entry Substrate X Oxidant (eq)
Py.9HF (eq)
Temp.
(oC)
Time (h)
Yieldb
1 72 H DBH (3) 6 25 2 80%
2 72 H DBH (3) 6 25 4 90%
3 72 H DBH (3) 6 35 1 85%
4 72 H DBH (3) 6 35 2 95%
5 73 Cl DBH (3) 6 25 1 85%
6 73 Cl DBH (3) 6 25 2 90%
7 73 Cl DBH (3) 6 35 1 95%
8 73 Cl DBH (2) 4 35 1 80%
9 74 Cl NBS (3) 6 35 4 25%
10 74 Cl NIS (3) 6 35 24 5%
11 75 OCH3 DBH (3) 6 35 16 70%
a Reactions were typically carried out in 0.5 mmol scale in CH2Cl2 b Yields were determined based on conversion of substrate to product as determined by 1H, 13C, 19F NMR and/or GC-MS.
Continuing with my optimization studies, I then treated ethyl 2-((4-
methoxyphenyl)thio)hexanoate 75 under the established conditions of DBH/Py.9HF
(3equiv./6equiv.) in CH2Cl2 at 35 oC. However, the reaction was very sluggish in nature
and gave ethyl 2,2-difluorohexanoate 76 in 70% NMR purity after 16 h of stirring (entry
11). Thus, it was concluded that 4-chlorophenyl thioethers reacted faster than the
corresponding phenyl and 4-methoxyphenyl thioethers, as noted by Haufe, et al.43
The variations in reactivity with change in the para substituent of the aromatic
group of the thioether can be explained by the plausible mechanism proposed by Haufe,
et al., for the desulfurization-difluorination reactions.43
40
Scheme 11. Proposed mechanism for the oxidative desulfurization-difluorination of alkyl-aryl thioethers by DBH/Py.9HF
The electron-donating groups (such as OMe) on the phenyl ring are thought to
stabilize the cationic charge on the resonance stabilized carbenium-sulfonium ion
intermediate (type C, Scheme 11) and thus promote the first fluorination step; while
electron-withdrawing substituents (such as Cl) are thought to ease the elimination of
arylsulfenyl bromide (from type E) in the last step and therefore promote the second
fluorination step. The proposed mechanism was supported by the observation that 4-
chlorophenyl thioether took half the time than the regular phenyl thioether (Table 6,
entries 1-4 vs 5-7); whereas 4-methoxyphenyl thioether reaction never went to
completion under analogous reaction conditions (entry 11).
Ester vs aldehyde vs ketone vs alkane
Next, I attempted to extend the desulfurization-difluorination of 2o alkyl-aryl
thioethers to substrates with other functional groups on the α-position of the thioether
moiety. Thus, the 2-((4-chlorophenyl)thio)-3-phenylpropanal (78)127 and 2-((4-
41
chlorophenyl)thio)-1-phenylpropan-1-one (79),128 which have a carbonyl functional
group on the α-carbon similar to an ester; and α-thioalkane (80), which does not have a
carbonyl group on the α-carbon; were prepared from their corresponding α-bromo
counterparts (Figure 20).
Figure 20. α-Thioaldehyde, α-thioketone and α-thioalkane substrates for attempted desulfurization-(di)fluorination reactions.
However, treatment of α-thioaldehyde 78 with DBH (3 equiv.) and Py.9HF (6
equiv.) in CH2Cl2 at 35 oC for 1 h, followed by aqueous workup showed complex 1H-
NMR spectrum and no signals in the 19F-NMR. And similar results were obtained when
α-thioketone 79 and α-thioalkane 80 were treated with DBH/Py.9HF under analogous
reaction conditions. No fluorination was observed with either of the three substrates even
after using excess amounts of DBH and Py.9HF, at higher temperatures and for
prolonged reaction times. Thus it was implied that my protocol for the deseulfurization-
difluorination of secondary/internal thioethers works for compounds with only ester
functional groups on the α-carbon.
As a result of functional group similarities between esters and lactones, I then
tried to extend the application of the desulfurization-defluorination methodology towards
the synthesis of gem-difluorinated lactone derivatives. Thus, treatment of 2-phenylthio-
5-pentanolide 81129 with DBH (4 equiv.) and Py.9HF (8 equiv.) in CH2Cl2 at 35 oC for 2
h, followed by aqueous workup gave yellow oil, which showed complex 1H-NMR
42
(Scheme 12). The 19F-NMR of the crude material showed triplets in the range of -100
ppm (J = 15.8 Hz), in agreement with the literature 19F-NMR peaks for gem-
difluorolactones,130 thus indicating the presence of the gem-difluorinated product 82.
Increase in duration of the reaction gave even more complex 19F-NMR spectrum.
Therefore, considering the volatile and unstable nature of the difluorolactones, I chose to
pursue less volatile 2,3-dideoxy-2-thioribonolactone precursors of type 83.
Scheme 12. Attempted synthesis of α,α-difluoro-δ-valerolactone 82.
Thus, treatment of (S)-(+)-2-phenylthio-4-benzyloxymethyl-4-butanolide 83131
with DBH (4 equiv.) and Py.9HF (8 equiv.) in CH2Cl2 at 35 oC for 16 h, followed by
aqueous workup showed complex TLC and multiple products on 1H-NMR (Scheme 13).
The 19F-NMR of the crude material showed multiplets in the region of -140 ppm and -
150 ppm. And HRMS showed m/z corresponding to α-fluorosulfide 84 and α-
fluorosulfoxide 85. This indicates that monofluorination occurred, albeit in very low
yield.
Scheme 13. Attempted synthesis of α,α-difluoro-γ-butyrolactone 86.
43
3.2.1.2. Oxidative desulfurization-fluorination approaches with uridine derivatives
Although my attempts in oxidative desulfurization-difluorination of 2,3-dideoxy-
2-(phenylthio)-2-S-ribonolactone precursor (83) were partially encouraging, I next
attempted analogous fluorination on 2'-S-aryl-2'-thiouridine analogues 89/90 and 2'-S-
aryl-2'-deoxy-2'-fluoro-2'-thiouridine analogue 93, since the substrates were readily
available for us (Scheme 14). The precursors 2'-S-(4-chlorophenyl)-2'-thiouridine 87 and
2'-S-(4-methoxyphenyl)-2'-thiouridine 88 were synthesized in three steps from uridine
following the well-established procedure of preparing 2,2'-anhydroarabinouridine132 from
uridine followed by ring opening with thiolates.133,134 The precursor 2'-S-(4-
chlorophenyl)-2'-thiouridine 87 was prepared by a slight modification of the published
procedure.121 The 4-chlorothiphenol reagent was stirred with PPh3 in DMF for 30 min at
room temperature before refluxing with 2,2'-anhydroarabinouridine, to reduce all the
possible disulfide to the free thiol. Subsequent protections of 3',5'-hydroxyl groups and/or
N3 with either acetyl or benzyl protection groups gave the desired 2'-S-aryl-2'-thiouridine
precursors 89/90 . The second class of substrates required for desulfurization-fluorination
reactions is α-fluoro thioether derivatives 93, which can be synthesized from thioether 90
by oxidation to the corresponding sulfoxides and subsequent fluorination of sulfoxides
92135,136 with DAST/SbCl3, as reported.134 In addition, sulfone precursors for reductive
desulfonylation-fluorination reactions were also readily prepared, following literature
protocols.134 Thus, oxidation of the 2'-S-aryl-2'-thiouridine precursors 89/90 with meta-
chloroperoxybenzoic acid (mCPBA) at ambient temperature gave the corresponding 2'-
deoxy-2'-arylsulfonyluridine derivatives 91/92. Analogously, 2'-deoxy-2'-fluoro-2'-(4-
methoxyphenyl)sulfonyluridine derivative 95 was obtained by treatment of 2'-deoxy-2'-
44
fluoro-2'-(4-methoxyphenyl)sulfonyluridine derivative 94 with mCPBA at ambient
temperature.
Scheme 14. Synthesis of 2'-arylthio substrates for the desulfurization-fluorination reactions.
Treatment of 3',5'-di-O-acetyl-2'-S-(4-chlorophenyl)-2'-thiouridine 89a with
DBH/Py.9HF at -78 oC, under Argon atmosphere for 2 h showed three new spots on TLC
(Scheme 15). Aqueous workup followed by column chromatography yielded three
different products in 41%, 18% and 30% respectively. The first product was established
to be 5-bromo-3',5'-di-O-acetyl-2'-S-(4-chlorophenyl)-2'-thiouridine (96a, 41%).
Signature peak (d or dd) for H5 at 5.67 ppm in 1H-NMR was missing and the
corresponding doublet for H6 (7.17 ppm) was collapsed to a singlet (6.98 ppm), implying
a C5-brominated product, which probably was caused by DBH. The remaining two
products were C5-brominated sulfoxide diastereoisomers [97 (R/S-S)]. Doublet of
doublets for H2' was found at 3.92 ppm for the faster moving sulfoxide and at 3.72 for
45
the slower moving isomer. In comparison, peak for H2' for the C5-brominated product 96
was at 3.47 ppm, and for the substrate 89a was at 3.78 ppm.
Fluorination attempts of 89a employing Py.9HF/NBS also gave the corresponding
5-brominated derivative 96a as major product (conditions not shown in scheme), while
no desired fluorination was observed (19F-NMR; data not shown). Based on these
findings, a novel and efficient protocol for C5-bromination of pyrimidine nucleosides and
C8-bromination of purine nucleosides with DBH and/or Lewis acids has been
developed.137 Analogous treatment of 89a with the recently developed NIS/DAST
system138,139 at rt as well as at 35 oC gave the corresponding 5-iodinated analogue 96b in
73% and 60% isolated yields respectively, with no indication of the fluorinated products.
Scheme 15. Oxidative desulfurization-difluorination of 2'-S-aryl-2'-thiouridine analogue 89a. Conditions A: DBH (1.1 eq), Py.9HF (excess), CH2Cl2, -78 oC, 2 h; Conditions B: NIS (3 eq), DAST (6 eq), CH2Cl2, 0 oC (6 h), then rt (32 h); Conditions C: NIS (3 eq), DAST (6 eq), CH2Cl2, 35 oC, 24 h.
Treatment of 89a with DBH (3 eq.)/Py.9HF (6 eq.)/CH2Cl2/35 oC/24 h gave a
complex reaction mixture showing formation of the 5-bromo-2'-α-fluorosulfoxide 99a
(<10% according to 1H, 19F NMR of the crude; data not shown; Figure 21). Changing the
acetyl protection group for a benzyl group did not improve the outcome of the reaction.
Treatment of the fully benzylated 89b with DBH (5 eq.)/Py.9HF (10 eq.)/CH2Cl2/35
46
oC/overnight gave a complex mixture indicating the formation of 5-bromo-2'-α-
fluorosulfide 98 or 5-bromo-2'-α-fluorosulfoxide 99b (1H, 19F-NMR of the crude).
Figure 21. Products for attempted fluorination of 2'-S-(4-chlorophenyl)-2'-thiouridine derivatives 89a and 89b
Similarly, treatment of fully benzylated 2'-S-(4-methoxyphenyl)-2'-thiouridine
90b with NIS (2.1 eq.)/Py.9HF (3 eq.) gave the corresponding 5-iodocounterpart (100,
29%) and the α-fluorothioether (94b, 12%) (Scheme 16). Analogous treatment of 90b
with the recently developed NIS/DAST system138,139 yielded the α-fluorothioether 94b in
44% isolated yield when 2.1 equiv. of NIS and 6 equiv. of DAST were used.
Interestingly, the 5-iodo derivative 100 was not observed with NIS/DAST system.
However, both the reactions failed to generate the desired geminal difluoro uridine
product. On the other hand, treatment of 90b with DBH (3 eq.)/Py.9HF (6 eq.)/35 oC
generated a complex reaction mixture Thus, it is noteworthy to conclude that NIS/DAST
system provided clean conversion of sulfide 90b to the mono-fluorinated analogue 94b in
respectable yield.
47
Scheme 16. Attempted oxidative desulfurization-fluorination of 2'-S-(4-methoxyphenyl)-2'-thiouridine analogue 90b. Conditions A: NIS (2.1 eq), Py.9HF (3 eq); Conditions B: NIS (2.1 eq), DAST (6 eq).
Next, the α-fluorothioether 94b, was also directly explored as substrate for the
second fluorination step leading to the expected gem-difluorouridine derivative.
Unfortunately, treatment of 94b with DBH/Py.9HF at -78 oC gave the corresponding 5-
brominated derivative as major product (101, 50%), with no indication of the geminal
difluoro product (Scheme 17). The introduction of the desired second fluorine atom was
unsuccessful even when excess reagents, higher temperatures, and longer reaction times
were employed.
O
SBnO
BnO
OCH3
N
N
O
O
Bn
O
SBnO
BnO
OCH3
N
N
O
O
BnBr
DBH (1.1 eq)Py.9HF (4.5 eq)
-78 oC, 3 h
94b 101 (50%)
CH2Cl2F F
Scheme 17. Attempted oxidative desulfurization-fluorination of 2'-fluoro-2'-S-(4-methoxyphenyl)-2'-thiouridine analogue 94b with DBH/Py.9HF. 3.2.2. Reductive desulfonylation-fluorination approaches
Since the oxidative desulfurization-difluorination approaches on 2'-arylthiouridine
derivatives were overshadowed by the formation of 5-halogenated byproducts and were
not very promising for the introduction of the geminal difluoro unit, I turned my attention
to reductive fluorination approaches of 2'-arylsulfonyluridine substrates. Reductive
desulfonylation-(di)fluorination protocols are to the best of my knowledge, unknown
processes, while chemical and electrochemical-induced reductive desulfonylation
reactions have been known for years.140-150 Chemical and electrochemical redox
48
strategies have rarely been reported in nucleoside series, except some reductive
dehalogenation or desulfonylation protocols en route to 2',3'-dideoxy-2',3'-didehydro
nucleosides with anti-HIV and anti-HBV properties.151-160
This part of the dissertation was completed in collaboration with Dr. Maurice
Medebielle of Université Claude Bernard Lyon 1, France; Dr. Gamal Giuglio and Dr.
Julie Broggi of Aix-Marseille Université, France.
3.2.2.1. Cyclic voltammetry studies of 2'-arylsulfonyl uridine derivatives
Initially, our collaborators performed cyclic voltammetry studies with various 2'-
deoxy-2'-S-(arylsulfonyl)-2'-thiouridine and 2'-deoxy-2'-fluoro-2'-S-(arylsulfonyl)-2'-
thiouridine substrates to find out the reduction potential at which the sulfone moiety
could be cleaved. Sulfone derivatives 92a and 92b (see Scheme 14 for structures), were
found to be reduced in two irreversible reduction steps in DMF containing 0.1 M n-
Bu4NPF6 on a glassy carbon electrode, at almost identical reduction potentials [- 2.73 V
and - 2.95 V for 92a; - 2.73 V and - 2.92 V for 92b (peak potentials at 0.2 V/s, Figure
22). For both substrates, the first irreversible reduction step (Epc1) corresponded to the
cleavage of the C2'-S bond with the expulsion of the p-methoxybenzenesulfinate,161 that
can be irreversibly oxidized at + 0.02 V (Epa2). The irreversibility of the first reduction
step (Epc1) is an indication that the first electron transfer is followed by a chemical
reaction producing a new product that is in turn reduced at a more negative potential
corresponding to the second reduction step (Epc2). Additional irreversible oxidation steps,
only observed after scanning first to reduction potentials and then switching to anodic
values, located at + 0.57 and + 0.81 V (92a, Epa3 and Epa4) and + 0.51 V (92b, Epa3) (peak
potentials at 0.2 V/s) were also noticed.
F=
st
B
m
ur
lo
nu
Figure 22. C= 2.75 mM, p
Cyclic
tructures) w
Both substra
meaning that
racil moiety
ocated at + 0
ucleosides a
yclic voltampink curve);
c voltammet
as also inve
tes are redu
the reductio
y reduction (
0.025 V only
are oxidized
mmetry of α-in DMF + n
try of 2'-α-f
estigated in D
uced irrever
on takes plac
(Figure 23).
y observed w
up to + 1.5 V
49
-sulfones 92a-Bu4NPF6 0
fluorosulfon
DMF in ord
rsibly at po
ce at relative
For both su
when scannin
V.
a (C = 2.25 .1M; v = 0.2
ne precursors
der to estima
otentials clo
ely high red
ubstrates an
ng in the cat
mM, blue cu2 V/s.
s 95a-b (see
ate their redu
ose to -2.67
duction poten
irreversible
athodic direc
urve) and 92
e Scheme 1
uction poten
V (vs Ag/
ntial not far
oxidation st
tion. None o
2b (C
4 for
ntials.
/Ag+)
from
tep is
of the
50
-4 -3 -2 -1 0 1 2-150
-100
-50
0
50
I (µ
A)
E (V)
Epc1
Epa1
Figure 23. Cyclic voltammetry of α-fluorosulfones 95a (2.64 mM) and 95b (2.91 mM) in DMF + NBu4PF6 0. 1 M; glassy carbon electrode.
The data obtained by cyclic voltammetry in DMF indicated that the cleavage of
the C2'-S bond, although possible will require (i) quite negative potential under
electrochemical activation and (ii) strong chemical reducing agents. Also glycosidic bond
cleavage may operate as well at such negative potentials as it is apparently the case for
substrates 92a-b. However, from cyclic voltammetry data, it is not obvious to conclude
what the possible reduction products resulting after the expulsion of the p-
methoxybenzenesulfinate are, at least on the time scale of the cyclic voltammetry.
Tentative isolation and characterization of the reductive products, either with the use of
chemical reducing agents or using electrochemical activation, was therefore necessary.
For 95a (--) Epc1
= - 2.66 V; Epa1
= + 0.025 V
For 95b (--) Epc1
= - 2.67 V; Epa1
= + 0.025 V
51
3.2.2.2. Reductive desulfonylation-fluorination studies of 2'-deoxy-2'-arylsulfonyl
uridine and 2'-deoxy-2'-fluoro-2'-arylsulfonyl uridine substrates
During preparation of sulfone substrates for the reductive fluorination approaches,
I observed an interesting result. Purification of 3',5'-di-O-acetyl-2'-deoxy-2'-[(4-
chlorophenyl)sulfonyl]uridine (91a) on silica gel column resulted in the overall
elimination of acetic acid to generate 2',3'-didehydro-2',3'-dideoxysulfone 102 as a major
product, which has same Rf value as starting material and thus inseparable (1.5:1 ratio of
102:91a; 1H-NMR) (Scheme 18). Structure of the product was confirmed by 1H-NMR
(doublet at 7.01 ppm with coupling constant of 1.9 Hz for H3') and also by comparing
spectral data with similar 2'-phenylselenonyl derivatives prepared by Chattopadhyaya, et
al.162 Generation of the elimination product 102 was indicative of the increased acidity of
C2'-H in C2'-sulfones 91a.
Scheme 18. Stability studies of sulfones 91a-b on silica gel chromatography or with treatment of base.
To avoid elimination of the acetate group from C3', a stable benzyl protection
group was employed (pKa of AcOH = 5 vs pKa of BnOH = 15). As expected, N3-benzyl-
3',5'-di-O-benzyl-2'-deoxy-2'-[(4-chlorophenyl)sulfonyl]uridine (91b) with benzyl
protecting group (poor leaving group) installed on 3',5' and N3 positions was stable
52
during silica gel column purification. Since the C2'-H in C2'-sulfones 91a-b is quite
acidic, I envisioned abstraction of C2'-proton with base followed by entrapment of the
resultant of C2'-carbanion with fluoronium ion (F+) source. However, treatment of
sulfone 91b with KH and Selectfluor in THF/DMF (-78 oC) led to the elimination of 3-N-
benzyluracil 103 (63%) via the glycosidic bond cleavage (Scheme 18). I also isolated 3,5-
di-O-benzyl-1,2-dideoxy-1,2-didehydro-2-[(4-chlorophenyl)sulfonyl]ribose (104; 71%)
from the reaction mixture, whose structure was established using spectroscopic data. The
singlet from the olefinic proton H1 at 7.4 ppm was diagnostic for the vinyl sulfone sugar
104. It was also found that treatment of sulfone 91b with base only, without Selectfluor,
also affected glycosidic bond cleavage to give 103 and 104, consistent with
base/Selectfluor system.
Since the base/F+ system proved to be too harsh for the sulfone substrates, I
imagined an alternative reduction pathway implying the selective cleavage of the sulfone
moiety using organic electron donors. Organic electron donors (OED) are powerful
reducing agents able to selectively cleave C-X (e.g., X = halogen) bonds by stepwise
transfer of one or two electron(s) under mild reaction conditions.163-170 Previous literature
studies report the efficient reduction of aryl iodides and bromides to aryl anions, as well
as the reductive cleavage of phenylalkylsulfones in excellent yields.171 With redox
potentials spanning from - 0.62 V to - 1.24 V vs SCE in DMF (~ - 0.93 V to - 1.55 V vs
Ag/Ag+ 0.01 M) (Figure 24), OED could allow us to reductively cleave the aryl-alkyl
sulfone in the 2'-sulfone precursors 92a-b and 2'-α-fluorosulfone precursors 95a-b.172
Indeed, in view of the high reduction potential of the arylsulfone moiety compared to
OED, transferring one electron would theoretically appear difficult. Nevertheless, as
53
pointed out by Murphy et al.,38 effective redox potentials of organic reducers in solution
are much higher than their thermodynamic redox potential determined by electrochemical
methods. It is explained by the formation of intimate charge-transfer complexes and ion
pairing that eases the electron transfer. Additional π-π stacking between the SED and
nucleoside may help to form intimate complex and facilitate the reduction. Hopefully, the
generated carbanion intermediate could then be used in electrophile trapping reactions.
Figure 24. Structures of organic electron donors used for the reductive desulfonylation.
Thus, treatment of sulfone 92a was treated with tetrakis(dimethylamino)ethylene
(TDAE) in DMF (-78 oC to rt) for 1.25 h followed by aqueous workup and silica gel
column chromatography gave furan derivative 105, the enol type (open chain sugar)
product 106, and the 3'-acetoxy elimination product 107 as major products (Scheme 19).
The anticipated reduction of sulfone moiety was never observed, indicating that the
TDAE initially acted as a base and then probably as a reducing agent. It is noteworthy to
mention that when 92a was treated with 4-dimethylamino pyridine (DMAP) in DMF at
room temperature, almost quantitative formation of 105 was produced, a result
supporting the basic pathway (not shown in scheme; data not given).
54
Analogous treatment of 92a with one equivalent of the more powerful Murphy’s
Super Electron Donor (N,N,N',N'-Tetramethyl-7,8-dihydro-6H-dipyrido[1,2-a;2',1'-
c][1,4]diazepine-2,12-diamine; SED)171 in DMF at ambient temperature for 15 min
yielded furan derivative 105 (95%). The nucleoside base uracil 105a was also identified
(but not isolated) by 1H-NMR in the aqueous phase along with other impurities.
Scheme 19. Reduction studies of acetyl protected sulfone substrates 91a with TDAE and Murphy’s SED.
Since the acetyl protection group in 92a is base labile and is prone to elimination
from the intermediate C2'-carbanion, I decided to use benzylated sulfone substrates.
However, treatment of N3-benzyl-3',5'-di-O-benzyl-2'-deoxy-2'-[(4-
chlorophenyl)sulfonyl]uridine (92b) with Murphy’s reagent in DMF for 15 min at
ambient temperature resulted in a similar, rapid glycosidic bond cleavage producing 3-N-
benzyluracil 103 (69%) and 3,5-di-O-benzyl-1,2-dideoxy-1,2-didehydro-2-[(4-
methoxyphenyl)sulfonyl]ribose (108; 70%) respectively (Scheme 20). This result is in
accordance with the result obtained when 92b was treated with base (Scheme 18) and
indicated that SED (in stoichiometric amount) was probably acting exclusively as base
55
and not as reducing agent. Also, analogous treatment of 92b with SED for 30 min at
room temperature generated uracil derivative 103 (in 86%), furan derivative 109 as a
mixture with vinyl sulfone 108.
Scheme 20. Reduction studies of benzyl protected sulfone substrate 91b with Murphy’s
SED.
Since the 2'-sulfone substrates were too labile under the desired reaction
conditions, the corresponding α-fluorosulfone substrates (95a-b, see Scheme 14 for
structures) were chosen for the further reductive-desulfonylation reactions. Initially,
stability of the acetyl protected 2'-(4-methoxyphenylsulfonyl)-2'-deoxy-2'-fluorouridine
95a towards tetrakis(dimethylamino)ethylene (TDAE) was evaluated. Thus, treatment of
95a (2'R/S, ~1:4.5) with 2 equiv. or 4 equiv. of TDAE in DMF at -20 °C for 1 h or at
room temperature for 3 h resulted in recovery of unchanged starting material 95a along
with 3'-deacetylated byproduct. Longer reaction time (12 h) and higher temperature (60
oC) produced only a mixture of mono- and di-deacetylated byproducts but no further
degradation or glycosidic bond cleavage were observed. Replacement of DMF with
MeCN gave similar results. Addition of TDAE at 0 oC or above did not cause the
reduction of the sulfonyl moiety, indicating that TDAE should be added at low
temperatures. Also, replacement of TDAE with other reducing agents such as samarium
iodide did not affect the reduction of sulfonyl unit.
56
On the other hand, treatment of α-fluorosulfone derivative 95a with 1 equiv. of
Murphy’s Super Electron Donor (SED) in DMF at rt or 120 oC, overnight gave the 3'-
deacetylated product (110) along with unchanged SM (Scheme 21). Similar results were
obtained with increased equiv. of SED (3 eq.) at rt. However, previously known
fluorovinyl compound 111173 was obtained in 46% yield when 95a was treated with 3 eq.
of SED (DMF, 120 oC, overnight) after acidic workup (10% HCl). The structure of 111
was established by 19F-NMR (δ -133.8 ppm, t, J = 4.6 Hz) and LC-MS [ESI+ m/z 271
(MH)+]. This result indicated that reductive cleavage of sulfone took place with excess
SED at high temperature. Noticeably, when 95a was treated with 4-dimethylamino
pyridine (DMAP) in DMF at room temperature, was recovered intact indicating that
base-induced pathway is not operating with such α-fluorosulfone.
Scheme 21. Stability studies of acetyl protected α-fluorosulfone 95a with Murphy’s SED.
To avoid the base-promoted elimination of the 3'-acetyl protection group, benzyl
protected α-fluorosulfone derivative 95b was employed to study the reduction of sulfone
with SED. However, treatment of N3-benzyl,3',5'-di-O-benzyl protected sulfone 95b
under analogous reaction conditions (3 eq. SED, DMF, 120 oC, 3 h) produced a complex
57
reaction mixture (Scheme 22). Careful silica gel column chromatography allowed for
separation of two major products, which are found to be glycosidic bond cleavage
products furan 112 and 3-N-benzyluracil 103. Trace amounts of sulfone cleavage product
113 was also isolated from the column. Intriguingly, in the furan derivative 112 sulfone
moiety was intact and elimination of fluorine was observed. Formation of 112 could have
been happened from either the reduction of glycosidic bond followed by base induced
fluoride elimination or vice versa.
Scheme 22. Stability studies of benzyl protected α-fluorosulfones 95b with Murphy’s SED
Analogous treatment of 95b with 3 equiv. of SED in DMF at room temperature
gave only trace amounts of 112 and 103 along with unchanged SM. Addition of degazed
water at 80°C to the crude, in order to favour the reduction product 113 (to enhance H-
abstraction), did not change the outcome of the reaction. The elimination product 112 and
3-N-benzyluracil 103 were obtained in 80% and 60% isolated yields respectively.
Next, in order to prepare gem-difluorouridine analogues, I attempted to insert a
second fluorine atom at C2'-position of 2'-arylsulfonyl-2'-deoxy-2'-fluorouridine
derivative 95a using reductive-desulfonylation/fluorination approach. If successful this
method could be expanded toward the synthesis of other 2'-alkyl-2'-deoxy-2'-fluoro
uridine nucleosides. Especially an intriguing target in that class of nucleosides would be
58
2'-C-methyl-2'-deoxy-2'-fluorouridine because of the potent anti-HCV activity possessed
by the corresponding cytidine analogue (PSI 6130).5
Scheme 23. Attempted synthesis of 2'-2''-difluorouridine by reductive desulfonylation-fluorination of 2'-arylsulfonyl-2'-deoxy-2'-fluorouridine 95a with TDAE/Selectrfluor
Since 95a was barely reactive towards TDAE at low temperatures with no
indication of the generation of putative C2' anion, I tried a combination of TDAE and UV
light activation to enhance the electron transfer. Thus, treatment of 95a with 5 equiv. of
TDAE at -60 °C and Selectfluor under uv light irradiation produced a mixture of 2',3'-
dideoxy-2',3'-didehydro-2'-fluorouridine 111 and 1-(2-deoxy-2-fluoro-β,D-
arabinofuranosyl)uracil 114 in addition to the acetyl deprotected byproducts (Scheme
23). Interestingly, no significant by-products resulting from the glycoside bond cleavage
was observed. It is noteworthy that when the TDAE was added at 0 °C or room
temperature, neither 111 nor 114 were formed, which means the TDAE did not act as a
reductant when added at higher remperatures. Furthermore, no formation of the target 2'-
deoxy-2',2'-difluorouridine derivative was observed, while the reduction of the employed
electrophilic fluorine reagents was detected.
59
Scheme 24. Reductive-desulfonylation/methylation of 2'-arylsulfonyl-2'-deoxy-2'-fluorouridine 95a with TDAE/MeI
Since fluoronium ion (F+) is the most difficult cation to work with, I chose to test
our hypothesis using CH3+. However, treatment of 95a with TDAE/MeI under analogous
reaction conditions yielded acetyl deprotected, N3-methylated analogue 115 as the major
product (Scheme 24).
3.3. One electron oxidation of gemcitabine and analogues
Our laboratory has a long history of studying the mechanism of ribonucleotide
reductase (RNR) enzyme via chemical modeling studies.174,175 In collaboration with Prof.
Stubbe of MIT, we have also been involved in studying mechanism-based inhibitors of
RNR by 2'-azido-2'-deoxynucleostides176,177 and probing the mechanism of RNR
catalyzed reaction with labeled natural substrates.178 Recently, in collaboration with Dr.
Sevilla’s group from Oakland University, we began investigating the generation of
radicals upon 1e- oxidation of various nucleoside substrates.179
Building on this interest, I wanted to explore the possibility of formation of sugar-
based radicals in the one-electron (1e-) oxidation of 2'-fluorocytidine analogues such as
2'-fluoro-2'-deoxycytidine (FdC, 38), 2'-deoxy-2',2''-difluorocytidine (dFdC, gemcitabine,
1), and 2'-deoxy-2'-fluoro-2'-C-methylcytidine (MeFdC, PSI 6130, 39) (Figure 25). Each
of the three substrates has different stereo and electronic properties which should interact
60
with the resultant radical differently. Comparing to 2'-fluoro-2'-deoxycytidine,
gemcitabine has an extra fluorine atom, which causes H3' to be more acidic because of its
negative inductive effect (-I). Thus I was also hoping that we might capture and analyze
the elusive C3' radical, postulated by Stubbe, et al.31 as a critical intermediate in the
reduction of RNA monomer to DNA monomer. The presence of a positive inductive
effect (+I) causing methyl group makes PSI 6130 a versatile candidate and it would be
interesting to study the influence of different substituents at 2'-position (H vs F vs CH3)
on radical generation. Electron spin resonance (ESR) spectroscopy was employed to
study the results generated during 1e- oxidation of the selected substrates.
O
FHO
HO
N
N
NH2
O
F
Gemcitabine1
O
FHO
HO
N
N
NH2
O
H
38
O
FHO
HO
N
N
NH2
O
PSI 613039
CH3
FdC
Figure 25. Selected substrates for 1e- oxidation studies
3.3.1. Detection of C2' and C3' sugar radicals
The ESR spectrum after γ-irradiation of 2'-deoxy-2'-fluorouridine 38 (7.5 M
LiCl/D2O sample of 2'-dC (2 mg/ml) at pH ca. 10) showed a central anisotropic doublet
(ca. 16 G) assigned to α proton at C5 in the 2'-F-dC pi-cation radical (C•+) 116 and is in
keeping with the coupling reported for the C5-H alpha proton coupling in the dC cation
radical in single crystals (Scheme 25).180
61
Scheme 25. One elctron oxidation of 2'-deoxy-2'-fluorocytidine
One-electron oxidation of gemcitabine 1 results in the formation of metastable pi-
cation radical 1a, which is unstable even at ca. 155 K (Scheme 26). The metastable C•+
1a quickly deprotonates at C3' in the sugar moiety producing C3'• 117 via a proton-
coupled electron-transfer (PCET) mechanism. The presence of 2nd fluorine atom at 2'
position may have increased the acidity of C3'H. Since the presence of one additional
fluorine led to such a drastic behavior, I was very interested to study one electron
oxidation of 2'-deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130; 39), which is very active
against HCV.181 PSI 6130 has a methyl group (+I) instead of a fluorine atom (-I).
Scheme 26. One electron oxidation of gemcitabine
The anti-HCV analogue PSI-6130 (39) also generated C3'-radical 39a via pi-
cation radical 118, upon γ-irradiation. But C3'-radical in PSI-6130 was unstable and
immediately converted to C2'-radical 119 via loss of hydrogen fluoride (Scheme 27). All
the results were in accordance with theoretical model studies and ESR spectroscopy.182
62
This is the first non-enzymatic example of C3'-radical studies on gemcitabine and PSI-
6130 and support the mechanism of inhibition of ribonucleotide reductase by these
compounds.
Scheme 27. One electron oxidation of PSI-6130
3.4. Synthesis and biological activity of 6-N-benzyl-7-deazapurine nucleoside
derivatives
3.4.1. Synthesis of 6-N-benzyl-7-deazapurine nucleosides
I wanted to prepare 6-N-benzylated 7-deazaadenosine analogues (122a-d) using
the Dimroth rearrangement, which is widely studied on various adenine nucleosides and
other heterocycles.115,183 Initial alkylation takes place at N1 when adenine nucleosides are
treated with alkyl/aryl halides to form an N6-centered cationic intermediate (120a;
Scheme 28). Treatment of the N1-alkylated intermediate 120a with base leads to ring-
opening, C-C bond rotation, and subsequent ring-closing to give the N6-alkylated
adenosine product 121. Interesting thing to note in the Dimroth rearrangement sequence
is that N1 of substrate becomes N6 in the product, which was established based on
isotope labeling studies.184,185
63
Scheme 28. General mechanism of Dimroth rearrangement on Adenosine184,185
Thus, treatment of tubercidin (19a) with benzyl bromide in DMF (48 h at 40 oC)
showed a baseline spot on TLC, which suggests ionic intermediate(s) (Scheme 29).
Evaporation of solvent; treatment of crude with acetone and ether; and vacuum filtration
gave a hygroscopic white precipitate. The ionic intermediate was immediately dissolved
in methanol and refluxed with dimethylamine (2 M solution in THF) (20 h at 65 oC).
TLC showed conversion of majority of the base line spot to a less polar product. Aqueous
workup followed by column chromatography gave 4-N-benzyltubericin 122a (67%).
Structure of the product was established by NMR and HRMS. For comparison, I
synthesized the previously known 6-N-benzyladenosine (121)115 from adenosine (120) by
following same reaction conditions (BnBr/DMF-Me2NH/MeOH). I noticed that the TLC
during the course of both the reactions was very similar. Therefore it is reasonable to
assume that initial alkylation occurred at N3 (same as ring nitrogen N1 in adenosine) in
64
tubercidin, and treatment of the 3-N-benzyl intermediate with Me2NH/THF in MeOH
resulted in Dimroth-type rearrangement to give 4-N-benzyltubercidin (122a).
Scheme 29. Synthesis of 4-N-benzylated tubercidin and sangivamycin by Dimroth rearrangement approach.
It is well-established that the 6-N-(4-nitrobenzyl) derivatives of adenosine and
other adenine nucleosides act as potent nucleoside transport inhibitors.119 To evaluate the
nucleoside transport inhibition properties in a related system, I endeavored to synthesize
4-N-(4-nitrobenzyl) derivatives of the 7-deaza antibiotics. Thus, following the protocol
for the synthesis of 122a, reaction of tubercidin 19a with 4-nitrobenzyl bromide (24 h at
80 oC) followed by treatment of the corresponding N-3 alkylated cationic intermediate
with Me2NH/MeOH produced 4-N-(4-nitrobenzyl)tubercidin (122b, 56%, Scheme 29).
An analogous base line spot was observed when sangivamycin (19b) was treated with 4-
nitrobenzyl bromide (48 h at 40 oC). Isolation of this ionic intermediate immediately
followed by treatment with Me2NH/MeOH yielded 4-N-(4-nitrobenzyl)sangivamycin
(122c, 45%). Structures of products 122b-c were established by NMR and elemental
analysis.
Since tubercidin and sangivamycin underwent Dimroth-type rearrangement when
treated with BnBr followed by Me2NH, I envisioned the synthesis of 4-N-(4-
65
nitrobenzyl)toyocamycin (122d) via similar synthetic pathway. However, upon treatment
of toyocamycin (19c) with 4-nitrobenzyl bromide (63 h at 40 oC), a major less polar spot
than substrate was observed on TLC, as opposed to a more polar spot in case of
tubercidin and sangivamycin (Scheme 30). Isolation of the new spot by column
chromatography and careful spectroscopic evaluation showed the compound as 4-N-(4-
nitrobenzyl)toyocamycin (122d; 46%). Since there was no N3-benzylated cationic
intermediate, there was no need for Me2NH/MeOH treatment. Therefore, a direct
alkylation of the exocyclic amine (N4) of toyocamycin was proposed for the formation of
122d.
Scheme 30. Synthesis of 4-N-(4-nitrobenzyl)toyocamycin by direct alkylation.
The direct alkylation of the exocyclic amino group of toyocamycin has not been
previously noted. However, direct alkylation on the exocyclic amino group of adenine
and guanine with quinone methides is known.186 The exocyclic amine nitrogen N4 may
have become more nucleophilic than the endocylcic nitrogen N3, as a result of the
electron withdrawing pull of the cyano group at C5. Another possibility could be that the
cylindrical cyano group might exist in synperiplanar orientation with the exocyclic amine
group, thereby diminishing the lone-pair donation on N4 toward N3 of the amidine,
which is part of the planar hetrocyclic system.187
66
Alternatively, the preparation of 6-N-(4-nitrobenzyl)tubercidin derivative (122b)
was envisioned based on synthesis of 6-fluorotubercidin analogue by diazotization-
fluorodediazoniation120,188,189 followed by SNAr displacement of 6-fluoro group with 4-
nitrobenzylamine.120,190 Thus, using standard protocol, tubercidin (19a) was treated with
acetic anhydride in pyridine to give 2',3',5'-tri-O-acetyltubercidin (123, 86%; Scheme 31).
The acetylated tubercidin (123) was then treated with sodium nitrite (NaNO2) in freshly
prepared ~55% HF-pyridine at -10 oC191,192 for 15 min to yield the protected 4-
fluorotubercidin 124 (82%), probably via diazotive-fluorodeamination. The mechanism
of this reaction is expected to be similar to ‘Balz-Schiemann’ type reaction for the
preparation of arylfluorides from arylamines using NaNO2 and 70% HF-pyridine.191
NaNO2 in the presence of acid (H+) generates nitrosonium ion (NO+) in situ and aryl-NH2
attacks the resultant NO+ to form diazonium ion 123a subsequently. Loss of N2 followed
by quenching of intermediate arylcation 123b with fluoride ion (F-) gives the product
arylfluoride 124. I observed that the concentration of HF was crucial in the preparation of
124. When commercial 70% HF-pyridine reagent was used, the reaction did not yield the
corresponding 4-fluoro product 124. This observation indicated that pH of the reaction,
which in turn depends on the percent of HF used, was critical for such diazotive-
fluorodeamination reactions.192
67
Scheme 31. Synthesis of 2',3',5'-tri-O-acetyl-4-fluorotubercidin (124) by diazotive-fluorodeamination.
Treatment of 4-fluorotubercidin derivative 124 with 4-nitrobenzylamine
hydrochloride in presence of trimethylamine (Et3N) in MeOH gave 2',3',5'-tri-O-acetyl-
4-N-(4-nitrobenzyl)tubercidin (125, 47%; Scheme 32). Trimethylamine converts 4-
nitrobenzylamine hydrochloride to 4-nitrobenzylamine in situ, and the free amine
subsequently displaces the fluoride ion, probably, by aromatic nucleophilic substitution
(SNAr).120 Acetyl deprotection of compound 125 using methanolic amonia (NH3/MeOH)
at ambient temperature gave 122b (85%; overall 32% yield). Out of the two methods for
the preparation of 122b, Dimroth alkylation pathway (56% yield) was better in terms of
number of steps and overall yield.
68
Scheme 32. Synthesis of 4-N-(4-nitrobenzyl)tubercidin (122b) from 2',3',5'-tri-O-acetyl-4-fluorotubercidin (124).
3.4.2. Biological activity of 6-N-benzyl-7-deazapurine nucleoside derivatives
Nucleoside transport inhibition activities were measured in Dr. James D. Young’s
laboratory of University of Alberta. While antiviral and anti-proliferation activities were
studied in Dr. Jan Balzarini’s laboratory of Rega Institute for Medical Research.
3.4.2.1. Nucleoside transport inhibition activity
Inhibition of the initial rate of 3H-uridine (20 µM) uptake by recombinant hENT1
produced in the Xenopus oocyte heterologous expression system was determined as
described previously.118 The 4-N-(4-nitrobenzyl)tubercidin (122b) and 4-N-(4-
nitrobenzyl)sangivamycin (122c) fully inhibited labelled uridine uptake, whereas 4-N-
benzyltubercidin (122a) and 4-N-(4-nitrobenzyl)toyocamycin (122d) analogues exhibited
weak inhibition (<45%). Dose-response evaluations indicated that sangivamycin
analogue 122c (IC50 = 123 ± 31 nM) was a better inhibitor of uridine transport by hENT1
than the tubercidin analogue 122b (IC50 > 1000 nM). However, the sangivamycin
analogue 122c was a weaker inhibitor of hENT1-mediated transport than 6-N-(4-
nitrobenzyl)adenosine, NBMPR, and other derivatives119 containing a nitrogen atom at
the 7-position in the adenine ring.
69
3.4.2.2. Antiviral activity
Table 7. Antiviral activity in human embryonic lung (HEL) cell cultures-part 1
Compd
EC50 (µM)*
HSV-1
(KOS)
HSV-2
(G)
Vaccinia virus
Vesicular stomatitis virus
HSV-1
TK - KOS ACV
122a >100 >100 >100 >100 >100
122b >100 >100 34 >100 >100
122c >100 >100 34 >100 >100
122d >100 >100 100 >100 >100
*Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU).
The 4-N-benzyl-7-deazapurine derivatives (122a-d) weakly inhibited Herpes
Simplex Virus (HSV; 1 & 2) and Vesicular stomatitis virus (EC50: >100 μM) (Table 7).
But Vaccinia virus was reasonably inhibited by 4-N-(4-nitrobenzyl)tubercidin (122b) and
4-N-(4-nitrobenzyl)sangivamycin (122c) (EC50: 34 μM) and to a weak extent by 4-N-
benzyltubercidin (122a) and 4-N-(4-nitrobenzyl)toyocmycin (122d) (EC50: >100 μM).
Cytomegalovirus (CMV) and Varicella Zoster Virus (VZV) were inhibited at ~20
µM concentrations by 4-N-benzyltubercidin (122a), 4-N-(4-nitrobenzyl)sangivamycin
(122c) and 4-N-(4-nitrobenzyl)sangivamycin (122d) analogues (Table 8). But 4-N-(4-
nitrobenzyl)tubercidin (122b) was not very potent (EC50: >100 μM).
70
Table 8. Antiviral activity human embryonic lung (HEL) cell cultures-part 2
Compd
EC50 (µM)*
CMV
(AD - 169 strain)
CMV
(Davis strain)
VZV
(TK + VZV strain)
VZV
(TK - VZV strain)
122a >20 >20 >20 >20
122b >100 >100 >100 >100
122c >20 >20 >20 >20
122d >20 >20 >20 >20
*Effective concentration required to reduce virus plaque formation by 50%. Virus input was 100 plaque forming units (PFU). 3.4.2.3. Inhibition of Cell Proliferation
Inhibition of the proliferation of murine leukemia (L1210) cells and human T-
lymphocyte (CEM), cervix carcinoma (HeLa), prostate cancer (PC-3), and kidney
carcinoma (Caki-1) cells by the 7-deazaadenine nucleoside analogues (122a-d) was
evaluated (Table 9). The 4-N-benzyltubercidin (122a) showed marked inhibition of the
proliferation of PC-3 cells (IC50: 0.92 µM) and HeLa cells (IC50: 7.4 µM) but no
significant activity was found with its nitrobenzyl analogue 122b (IC50: >50 µM). The 4-
N-(4-nitrobenzyl)sangivamycin (122c) and 4-N-(4-nitrobenzyl)toyocamycin (122d)
analogues inhibited proliferation of L1210, HeLa, and PC-3 cells at ∼0.9–9.4 µM with
the sangivamycin analogue 122c showing more potent effects (0.92–3.4 µM). It is
intriguing that cytostatic activity of the 6-N-benzyl-7-deazapurine analogues was highly
dependent on the nature of the tumor cell lines. Proliferation of HeLa and PC-3 tumor
cells was highly inhibited by 122a, 122c, and 122d whereas proliferation of L1210 cells
71
was sensitive only to 122c and 122d. The CEM and Caki-1 cells were weakly sensitive to
the antiproliferative effects of any of the 6-N-benzyl-7-deazapurine compounds 122a–
122d.
Table 9. Inhibitory effects on the proliferation of cells in culture
Compd IC50 (µM)*
L1210 CEM HeLa PC-3 Caki-1
122a 172 ± 47 182 ± 20 7.4 ± 2.2 0.92 ± 0.67 116 ± 23
122b 125 ± 28 ≥250 143 ± 4 76 ± 10 132 ± 30
122c 0.92 ± 0.04 115 ± 28 1.8 ± 0.2 3.4 ± 0.9 116 ± 23
122d 5.5 ± 1.5 109 ± 16 9.4 ± 3.0 5.6 ± 3.3 98 ± 10
* Half-maximal inhibitory concentration (IC50); data represent the mean_ SD of at least n=2–3 independent experiments; Cell lines: murine leukemia (L1210); human CD4+ T-lymphocytes (CEM); human cervix carcinoma (HeLa); human prostate adenocarcinoma (PC-3); human kidney carcinoma (Caki-1).
3.5. C7- and C8-modified-7-deazapurine nucleoside derivatives
Building upon my interest in working with 7-deazaadenosine nucleosides, I
envisioned the synthesis of various 7-substituted or 8-substituted 7-deazaadenosine
analogues, which might have fluorescent properties.
3.5.1. Strain promoted click reactions of 8-azido-7-deazanucleosides with
cyclooctynes
Initially, I imagined the conversion of 8-halo-7-deazapurines to the corresponding
8-azido-7-deazapurines, which could serve as substrates for Strain-promoted Azide-
Alkyne Cycloaddition reactions (SPAAC, Cu-free click chemistry) with
72
cyclooctynes.193,194 I expect that the resulting triazolyl products might have fluorescent
properties due to increased conjugation in the 7-deazapurine base part. The Cu-free click
chemistry is a rapidly growing field, with various applications for in vivo imaging
emerging continuously over the past few years.193,195-199
Thus, 8-Bromotubercidin (126) was prepared by treatment of tubercidin 19a with
N-bromosuccinimide in presence of potassium acetate (KOAc) in DMF at ambient
temperature (30 min), as reported in literature.200 I envisioned the preparation of 8-azido-
7-deazaadenosine derivatives (127-129, Scheme 33) by nucleophilic aromatic
substitution of 8-bromo-7-deazaadenosine derivatives (55, 56 and 126) with sodium
azide. However, Attempts to synthesize 8-azidotubercidin via nucleophilic aromatic
substitution of 126 with sodium azide were unsuccessful, even after using harsh
conditions (NaN3 (1-5 eq), DMF, rt-153 °C, N2, 24 h; NaN3 (3 eq), TsOH (3 eq), EtOH,
reflux, 24h). Lack of reaction is possibly a consequence of the low electron deficiency of
the pyrrole ring in tubercidin 19a when compared to that of other 7-deazanucleosides
19b-c. To explain in other words, C8-bromination probably proceeds via electrophilic
aromatic substitution, whereas C8-azidation is expected to proceed via nucleophilic
aromatic substitution. I envisioned the presence of electron withdrawing groups in the
pyrrole ring on 8-bromo-7-deaza nucleosides will enhance the electron deficiency, there
by promoting nucleophilic displacement of bromide with azide. So I chose to pursue 8-
bromotoyocamycin and 8-bromosangivamycin substrates, both of which have an electron
withdrawing substituent on the 7-position of the pyrrole ring.
73
Scheme 33. Synthesis of 8-bromo-7-deazaadenosine and 8-azidotoyocamycin derivatives.
Treatment of 8-bromosangivamycin 56 with NaN3 in DMF at 70 ºC for 16 hrs
gave a slightly more polar product, which was thought to be 8-azidosangivamycin. UV
spectra of the reactions always showed at least 12 nm bathochromic shift. However, ions
corresponding to neither product (128) nor substrate (56) were detected in the high-
resolution mass spectroscopy (HRMS). Instead, peaks at m/z 258 and 317 were observed,
which were unable to be identified with a list of possible products. Similar results were
obtained when DMSO or MeOH were used as the reaction solvents.
Next, I synthesized 8-azidotoyocamycin 129 by treating 8-bromotoyocamycin 55
with NaN3 in DMF at room temperature for 16 h. This transformation was light and,
possibly, heat sensitive requiring the reaction, purification, and characterization to be
carried out in the dark with limited heating to give 8-azidotoyocamycin (129; 46%). The
light sensitivity of the product also limited us in recording the analytical data for the
compound. Moreover, this azidation (55 to 129) was unsuccessful in MeOH or water,
indicating that polar aprotic solvents are probably the best solvents for these types of
aromatic nucleophilic substitution reactions.
74
Scheme 34. Click reaction between 8-azidotoyocamycin and cyclooctynes 130 and 131. Reagents and conditions: g) Cyclooctyne 130 or 131 (1 eq), ACN:H2O (3:1), rt, 4 h.
Having 8-azidotoyocamycin (129) in hand, my next goal was to see if the SPAAC
reactions would take place between 129 and cyclooctynes. Thus, treatment of 129 with
symmetrically fused cyclopropyl cyclooctyne 130 in an aqueous solution of acetonitrile
(ACN) at ambient temperature (4 h) gave the corresponding triazolyl product 132 (59%,
Scheme 34). Analogous reaction of 129 with a strain modulated dibenzylcyclooctyne 131
produced triazole 133 (47%). These preliminary results indicated that the SPAAC
reactions were taking place as expected. With the extended conjugation in the
deazapurine part, I expect the triazole products to have fluorescence properties and that
could lead to various interesting biological applications.
3.5.1. Attempted synthesis of 8-alkynyl-7-deazanucleosides via Sonagashira reaction
Since the attempted synthesis of 8-azido analogues of tubercidin and
sangivamycin were unsuccessful, I envisioned the synthesis of 8-vinylazido derivatives
(136) from the corresponding 8-alkynyl-7-dezapurine derivatives (135), which in trun
75
could be prepared in two steps via Sonagashira coupling of 8-halo-7-deazapurines (55, 56
and 126) with trimethylsilylacetylene (Scheme 35). The vinylazides 136 could be used as
substrates for strain-promoted click reactions to give the corresponding triazolyl products
(137). Also, the crucial 8-alkynyl derivative 135 could also be converted to the
corresponding halovinyl sulfone and β-keto sulfones (not shown in scheme). This part of
the dissertation is envisioned based on recent literature relating to transformations of
alkynes to the corresponding (i) halovinyl sulfones;201-203 (ii) β-keto sulfones;204 and (iii)
vinylazides.205
Scheme 35. Proposed scheme for the synthesis and subsequent transformations of 8-alkynyl-7-deazapurine nucleoside analogues 135.
Initially, treatment of 8-bromotubercidin (126) with trimethylsilylacetylene
(TMSA) in the presence of trimethylamine (Et3N) and catalytic amounts of
bis(triphenylphosphine)palladium dichloride [(PPh3)2PdCl2] and copper iodide (CuI)
76
(anhydrous DMF, 110 oC, sealed flask) showed unchanged starting material as major
product (Scheme 35; Table 10, entry 1). Doubling the amounts of CuI and Et3N did not
change the outcome of the reaction (entry 2).
Table 10. Reaction conditions for the attempted Sonagashira cross-coupling reactions on 8-bromo tubercidin and toyocamycina
Entry Substrate (PPh3)2PdCl2
(mol %)
CuI
(mol %) Et3N TMSA Temp. Time
1 126 3% 9% Excess 3 eq. 110 oC 48 h
2 126 3% 15% Excess 6 eq. 110 oC 48 h
3 126 10% 20% 4 eq. 3 eq. 50 oC 16 h
4 126 15% 30% 11 eq. 10 eq. 50 oC 5 h
5 55 1% 2% 7 eq. 1.2 eq. 80 oC 48 h
6 55 10% 20% 5 eq. 3 eq. 50 oC 24 h
7 55 20% 10% 5 eq. 3 eq. 50 oC 24 h
8 55 15% 30% 14 eq. 10 eq. 50 oC 22 h aReactions were typically performed on 0.1 mmol scale in anhydrous DMF. Reaction progress was monitored by TLC.
Treatment of 126 with 0.1 euqiv. of Pd and 0.2 equiv. Cu catalysts under
analogous reaction conditions showed a minor less polar spot on TLC (entry 3).
Encouraged by the observation, I treated 8-bromotubercidin 126 with 0.15 equiv. of Pd
catalyst, 0.3 equiv. of Cu catalyst, 11 equiv. of Et3N, and 10 equiv. of TMSA in
anhydrous DMF for 5 hrs at 50 oC. TLC of the crude reaction mixture was complex, but
there was a major less polar spot (~50% in comparison with unchanged substrate) (entry
4). 1H-NMR of the isolated material showed ~55:45 ratio of substrate to product
(126:134a). The mixture of products was then treated with tetrabutylammonium fluoride
(TBAF) and the major product was isolated by column chromatography. But 1H-NMR of
77
the isolated spot showed absence of the characteristic acetylenic proton peak and was
very similar to the 1H-NMR of 8-bromotubercidin 126, and no signals for the 8-alkynyl
product 134a were observed. Thus, it can be concluded that Sonagashira cross-coupling
of 8-bromotubercidin with trimethylsilylacetylene is a low-yielding reaction.
Because of the ease of preparation and availability of precursors, I used 8-
halotoyocamycin derivatives for initial Sonagashira reactions before proceeding to 8-
halosonagashira substrates. However, analogous treatment of 8-bromotoyocamycin (55)
with trimethylsilylacetylene [(PPh3)2PdCl2/CuI/Et3N/DMF/40 oC) also gave unchanged
starting material as major product, even after varying the reaction conditions (Scheme 35;
Table 10, entries 5-8).
To simplify the purification process, I decided to use protected 8-halo-7-
deazapurine derivatives as substrates. Thus, 2',3',5'-tri-O-acetyltoyocamycin (138) was
prepared in 97% isolated yield by treating toyocamycin (19c) with Ac2O/Py (Scheme 36).
Treatment of 2',3',5'-tri-O-acetyltoyocamycin (138) with DBH (1.1 eq., CH2Cl2, 18 h)
gave the corresponding 8-bromo-2',3',5'-tri-O-acetyltoyocamycin (139) in 79% isolated
yield. However, treatment of 139 (107 mg) with 0.02 equiv. of Pd catalyst, 0.1 equiv. of
Cu catalyst, 0.1 equiv. of Et3N, and 4 equiv. of TMSA in anhydrous DMF for 19 h at 40
oC gave a complex reaction mixture. Silica gel column chromatography gave three major
fractions. The NMR analysis showed the first two products to be unchanged starting
material 139 (27 mg, 25%), and debrominated product 138 (7 mg, 8%). The third fraction
(4 mg) was more polar than the substrate on TLC, was blue on TLC plate under 365 nm
lamp. But 1H-NMR of this material did not have characteristic product peaks and
structure of this blue spot could not be determined.
78
Scheme 36. Attempted synthesis of 8-alkynyltoyocamycin derivative 140 via sonagashira coupling.
As iodo is a better substrate than bromo in Sonagashira coupling, I envisioned the
use of 8-iodo-7-deazapurine analogues as substrates. Thus, toyocamycin 19c was
converted to the corresponding tri-O-(tert-butyldimethylsilyl) protected counterpart 141
using standard procedures (TBDMSCl/Imidazole/DMF/35 oC; 58%) (Scheme 37). The
reaction was sluggish and required longer hours of stirring and more equivalents of
reagents than usual. Treatment of silylated toyocamycin 141 with Lithium
diisopropylamide (LDA, 5 equiv.) and iodine (I2, 1.5 equiv.) in dry THF [-78 oC (2 h) to
ambient (48 h)] gave the corresponding 8-iodotoyocamycin analogue 142 in 16% isolated
yield. Unchanged substrate 141 was also isolated form the column (58%). However,
treatment of 8-iodo analogue 142 with trimethylsilylacetylene, (PPh3)2PdCl2 (10% mol),
CuI (20% mol), Et3N (excess), TMSA (4 equiv.) in DMF at 40 oC for 48 h gave
unchanged starting material 142 as major product even after for 2 days. Strangely, no
trace of deiodinated product 141 or acetylenic product 143 was observed on TLC.
79
Scheme 37. Attempted synthesis of 8-alkynyltoyocamycin derivative 143 via sonagashira coupling.
Considering the cost of 7-deazapurine substrates, I stopped proceeding further in
this direction.
3.5.2. Iodine catalyzed direct C-H activation of 7-deazapurine nucleosides
Since I was unable to synthesize the 8-azido or the corresponding vinylazide
analogues of tubercidin and sangivamycin, I explored other possibilities for incorporation
of an aromatic moiety (e.g. triazolyl) based on direct on C-H activation.206 Incidentally, a
recent research paper by Dr. Yotphan, et al. described iodine-catalyzed direct activation
of indoles with azoles mediated by tert-butylhydroperoxide (TBHP) (Scheme 38).207
Scheme 38. Iodine catalyzed oxidative cross-coupling of Indoles and Azoles.207
Because tubercidin 19a is structurally similar to indole, I imagined oxidative
cross-coupling of tubercidin with triazoles to generate the corresponding C7 and/or C8
triazolyl substituted analogues. Thus, initial treatment of tubercidin 19a with
benzotriazole (2 equiv.), I2 (0.2 equiv.), and TBHP (5-6 M solution in decane, 1 equiv.) in
80
anhydrous DMF at ambient temperature after 7 h showed unchanged tubercidin as the
major product on TLC (Scheme 39). No product formation was observed even after
stirring the reaction mixture at 40 oC for 7 hrs. Additional I2 (0.2 equiv.), and TBHP (1
equiv.) were added and the reaction mixture was stirred at 40 oC for 48 h, by which time
the TLC showed ~15% conversion to a more polar product. Analogous treatment of
tubercidin 19a with benzotriazole (2 equiv.), I2 (0.4 equiv.), and TBHP (2 equiv.) in
anhydrous DMF at 35 oC for 72 h also showed ~10% conversion to a less polar product.
Additional TBHP (1 equiv.) was added and the reaction mixture was stirred at 35 oC for
24 h (total: 96 h), by which time the TLC showed ~30% conversion to a more polar
product. The new spot was isolated by silica gel chromatography in 18% yield and was
single product on 1H, 13C NMR. Comparison of 1H-NMR data with substrate revealed
the following: (i) singlet for H2 was still present (shifted to 8.22 ppm from 8.05 ppm); (ii)
Doublet for H8 (7.35 ppm) collapsed to a singlet (7.04 ppm); and (ii) doublet for H7 (at
7.6 ppm) is absent. This confirms the structure of the product to be 7-
(benzotriazolyl)tubercidin 144.
Scheme 39. Iodine catalyzed oxidative cross-coupling of tubercidin and benzotrizole.
Fluorescence analysis of the triazolyl compound 144 showed emission at 420 nm
with the best quantum yield being ~0.002 units (in MeOH). The fluorescence is stronger
in basic pH between 7 and 12 and at pH ~2, it is non-fluorescent. The average lifetime is
81
about 3.8 ns with two well-defined lifetimes of 1 ns (10%) and 4.1 ns (90%). However,
the compound seems to be highly sensitive to UV light, since increase in fluorescence
upon irradiation with 280 nm light was observed.
Conversely, treatment of acetyl-protected tubercidin (123) with I2 (0.2 eq.), TBHP
(1eq.), in DMF at room temperature for 12 h gave a mixture of three products (Scheme
40). TLC of the reaction mixture showed ~30% conversion to a less polar spot. Isolation
of the new spot by column chromatography followed by 1H-NMR analysis showed the
spot as a mixture of three different products (19% yield, 60:25:15 mixture). It is
suspected that the mixture of products are 7-substituted analogue 145, 8-substituted
counterpart 146 and probably 7,8-dibenzotriazolyltubercidin (not shown in scheme).
Scheme 40. Iodine catalyzed oxidative cross-coupling of acetylated tubercidin and benzotriazole.
In addition, treatment of sangivamycin (19b) with benzotriazole (2 eq.), I2 (0.4
eq.), TBHP (2 eq.), in anhydrous DMF at 120 oC for 24 h showed unchanged starting
material on TLC (Scheme 41). On the other hand, treatment of toyocamycin (19c) with
benzotriazole (2 eq.), I2 (0.4 eq.), TBHP (2eq.), in anhydrous DMF at 80 oC for 48 hrs
resulted in 30% conversion to new spot on TLC. The isolated spot was not the expected
product 147c and exact structure could not be established by spectroscopic data. These
82
results reiterate the literature reports that indoles with electron deficient pyrrole rings
react sluggishly with azoles.207
Scheme 41. Attempted synthesis of iodine catalyzed oxidative cross-coupling of sangivamycin or toyocamycin and benzotriazole.
4. EXPERIMENTAL SECTION
The 1H (400 MHz), 13C (100.6 MHz), and 19F (376 MHz) NMR spectra were
recorded at ambient temperature in solutions of CDCl3 or DMSO-d6. Reaction progress
was monitored by TLC on Merck Kieselgel 60-F254 sheets with product detection by 254-
nm light. Products were purified by column chromatography using Merck Kiselgel 60
(230-400 mesh) or by automated flash chromatography using a CombiFlash system. UV
spectra were recorded with a Varian Cary 100 Bio UV-visible spectrophotometer.
Reagent grade chemicals were used and solvents were dried by reflux and distillation
from CaH2 under N2 unless otherwise specified, and an atmosphere of N2 was used for
reactions. Purity of the synthesized compounds was determined to be ≥95% by elemental
analysis (C, H, N) and/or HPLC on Phenomenex Gemini RP-C18 column with
CH3CN/H2O solvent system as mobile phase. Elemental analyses were performed by
Galbraith Laboratories, Knoxville, TN. Mass spectra (MS) were obtained with
atmospheric pressure chemical ionization (APCI) technique or electro-spray ionization
83
(ESI) techniques. High-resolution mass spectra were obtained in ESI-TOF mode unless
otherwise noted.
Chemical shifts (δ) are reported in parts per million (ppm) referenced to the
residual solvent peak, and coupling constants (J) are given in Hertz (Hz). Multiplicity is
reported using standard abbreviations: singlet (s); doublet (d); triplet (t); multiplet (m);
broad (br); inverted commas indicate observed multiplicity. UV spectra were recorded
with a Varian Cary 100 Bio UV-visible spectrophotometer.
Some of the data were reprinted with permission from the following papers:
(i) Adhikary, Amitava; Kumar, Anil; Rayala, Ramanjaneyulu; Hindi, Ragda M.;
Adhikary, Ananya; Wnuk, Stanislaw F.; and Sevilla, Michael D., “One-electron
oxidation of Gemcitabine and analogs: Mechanism of formation of C3′ and C2′
sugar radicals”, J. Am. Chem. Soc. 2014, 136 (44), 15646−15653. Copyright ©
2014, American Chemical Society.
(ii) Rayala, Ramanjaneyulu; Theard, Patricia; Ortiz, Heysell; Yao, Sylvia; Young,
James D.; Balzarini, Jan; Robins, Morris J., Wnuk, Stanislaw F., “Synthesis of
Purine and 7-Deazapurine Nucleoside Analogues of 6-N-(4-
Nitrobenzyl)adenosine; Inhibition of Nucleoside Transport and Proliferation of
Cancer Cells”, ChemMedChem 2014, 9, 2186-2192. Copyright © 2014 WILEY-
VCH Verlag GmbH & Co. KGaA, Weinheim.
(iii) Rayala, Ramanjaneyulu; Wnuk, Stanislaw F., “Bromination at C-5 of pyrimidine
and C-8 of purine nucleosides with 1,3-dibromo-5,5-dimethylhydantoin”,
Tetrahedron Letters 2012, 53 (26), 3333-3336. Copyright © 2012, Elsevier Ltd.
Cytostatic activity assays
84
All assays were performed in 96-well microtiter plates. To each well of 200 µL were
added (5−7.5) × 104 tumor cells and a given amount of the test compound. The cells were
allowed to proliferate for 48 h (murine leukemia L1210), 72 h (human lymphocytic
CEM), 96 h (human cervix carcinoma HeLa), 144 h (human prostate cancer PC-3), or
168 h (human kidney Caki-1) at 37 °C in a humidified CO2-controlled atmosphere. At the
end of the incubation period, the cells were counted in a Coulter counter. The IC50 (50%
inhibitory concentration) was defined as the concentration of the compound that inhibited
cell proliferation by 50%.
General procedure for the bromination of protected nucleosides. DBH and/or
TMSOTf were added to a stirred solution of substrate in CH2Cl2. The resulting brownish-
orange solution was stirred at room temperature for time given in the corresponding table
(Tables 3, 4, and 5; Section 3.1.) or until TLC showed absence of starting material and
formation of less polar product. The reaction mixture was diluted with CHCl3 and was
washed with saturated NaHCO3/H2O and brine. The organic layer was dried (MgSO4)
and concentrated in vacuo to yield the corresponding brominated product as a colorless
foam with purity over 98% (1H NMR).
General procedure for the bromination of unprotected nucleosides.
Procedure A. DBH was added to a stirred solution of substrate in DMF. The resulting
pale-yellow solution was stirred at room temperature for time given in the corresponding
table (Tables 3, 4, and 5; Section 3.1.) or until TLC showed absence of starting material
and formation of less polar product. Volatiles were evaporated and the residue was co-
evaporated with MeCN. The resulting pale solid was crystallized or purified by column
chromatography to give the corresponding brominated product as colorless crystals.
85
Procedure B. DBH was added to a stirred suspension/solution of substrate in MeOH.
The resulting pale solution was stirred at room temperature for time given in the
corresponding table (Tables 3, 4, and 5; Section 3.1.) or until TLC showed absence of
starting material and formation of less polar product. Products were isolated by (a)
filtration, if the products precipitated from the reaction mixture; (b) if no was precipitate
observed, then volatiles were evaporated and the residue was crystallized or column
chromatographed to give the corresponding brominated product as colorless crystals. The
isolated brominated products were usually >98% pure by 1H-NMR.
5-Bromo-2',3',5'-tri-O-acetyluridine (46a)64. UV (MeOH) λmax 275 nm, λmin 240 nm; 1H
NMR (CDCl3) δ 9.75 (br s, 1H, NH), 7.82 (s, 1H, H6), 6.06 (d, J = 4.7 Hz, 1H, H1'),
5.34-5.29 (m, 2H, H2', H3'), 4.38-4.30 (m, 3H, H4', H5', H5''), 2.18 (s, 3H, Ac), 2.10 (s,
3H, Ac), 2.08 (s, 3H, Ac).
5-Bromo-1-(2,3,5-tri-O-acetyl-β-D-arabinofuranosyl)uracil (46b).208 UV (MeOH)
λmax 276 nm, λmin 239 nm; 1H NMR (CDCl3) δ 9.33 (br s, 1H, NH), 7.77 (s, 1H, H6),
6.22 (d, J = 4.1 Hz, 1H, H1'), 5.35 (dd, J = 3.7, 4.1 Hz, 1H, H2'), 5.04 (“q”, J = 1.9 Hz,
1H, H3'), 4.38 (dd, J = 5.8, 12.1 Hz, 1H, H5'), 4.32 (dd, J = 3.9, 12.1 Hz, 1H, H5''), 4.16-
4.12 (m, 1H, H4'), 2.09 (s, 3H, Ac), 2.07 (s, 3H, Ac), 1.98 (s, 3H, Ac); 13C NMR
(CDCl3): δ 170.5, 169.6, 168.6 (3 x Ac), 158.6 (C4), 149.2 (C2), 139.8 (C6), 96.3 (C5 ),
84.4 (C1'), 80.7 (C4'), 76.1 (C3'), 74.4 (C2'), 62.5 (C5'), 20.8, 20.6, 20.4 (3 × Ac); MS
(ESI) m/z 447 (100, [79Br], MH-), 449 (98, [81Br], MH-).
5-Bromo-3',5'-di-O-acetyl-2'-deoxy-uridine (46c)64. UV (MeOH) λmax 277 nm, λmin 241
nm; 1H NMR (CDCl3) δ 9.59 (br s, 1H, NH), 7.89 (s, 1H, H6), 6.29 (“dd”, J = 5.8, 8.0
Hz, 1H, H1'), 5.24-5.21 (m, 1H, H3'), 4.40 (dd, J = 3.1, 12.2 Hz, 1H, H5'), 4.34-4.28 (m,
86
2H, H4', H5''), 2.54 (ddd, J = 2.3, 5.8, 14.3 Hz, 1H, H2'), 2.23-2.15 (m, 1H, H2''), 2.17 (s,
3H, Ac), 2.11 (s, 3H, Ac).
5-Bromouridine (46d)64. UV (MeOH) λmax 279 nm, λmin 241 nm; 1H NMR (DMSO-d6) δ
11.81 (br s, 1H, NH), 8.48 (s, 1H, H6), 5.73 (d, J = 4.5 Hz, 1H, H1'), 5.42 (d, J = 5.3 Hz,
1H, 2'OH), 5.27 (t, J = 4.7 Hz , 1H, 5'OH), 5.07 (d, J = 5.4 Hz, 1H, 3'OH), 4.04 (q, J =
4.9 Hz, 1H, H2'), 3.98 (q, J = 5.0 Hz, 1H, H3'), 3.88-3.85 (m, 1H, H4'), 3.71-3.66 (m, 1H,
H5'), 3.60-3.55 (m, 1H, H5'').
5-Bromo-1-(β-D-arabinofuranosyl)uracil (46e)209. UV (MeOH) λmax 280 nm, λmin 242
nm; 1H NMR (DMSO-d6) δ 11.82 (br s, 1H, NH), 8.07 (s, 1H, H6), 5.96 (d, J = 4.6 Hz,
1H, H1'), 5.63 (d, J = 5.3 Hz, 1H, 2'OH), 5.48 (d, J = 4.5 Hz , 1H, 3'OH), 5.17 (t, J = 5.4
Hz, 1H, 5'OH), 4.03 (“q”, J = 4.5 Hz, 1H, H2'), 3.91 (q, J = 4.2 Hz, 1H, H3'), 3.74 (“q”, J
= 4.5 Hz, 1H, H4'), 3.66-3.55 (m, 1H, H5', H5'').
5-Bromo-2'-deoxyuridine (46f)64. UV (MeOH) λmax 279 nm, λmin 241 nm; 1H NMR
(DMSO-d6) δ 11.79 (br s, 1H, NH), 8.39 (s, 1H, H6), 6.10 (t, J = 6.5 Hz, 1H, H1'), 5.25
(d, J = 4.3 Hz , 1H, 3'OH), 5.17 (t, J = 4.8 Hz, 1H, 5'OH), 4.26-4.22 (m, 1H, H3'), 3.79
(q, J = 3.2 Hz, 1H, H4'), 3.66-3.54 (m, 2H, H5', H5''), 2.18-2.08 (m, 2H, H2', H2'').
5-Bromocytidine (47b)62. UV (MeOH) λmax 289 nm, λmin 265 nm; 1H NMR (DMSO-d6)
δ 8.39 (s, 1H, H6), 7.83 (br s, 1H, NH), 6.99 (br s, 1H, NH), 5.71 (d, J = 3.2 Hz, 1H,
H1'), 5.37 (d, J = 5.0 Hz, 1H, 2'OH), 5.23 (t, J = 4.8 Hz , 1H, 5'OH), 5.00 (d, J = 5.4 Hz,
1H, 3'OH), 3.98-3.92 (m, 1H, H2'), 3.85-3.83 (m, 1H, H3'), 3.70 (ddd, J = 2.7, 4.8, 12.1
Hz, 1H, H5'), 3.56 (ddd, J = 2.5, 4.9, 12.1 Hz, 1H, H5'').
4-N-Benzoyl-5-bromocytidine (48b). mp 193-195 oC; UV (MeOH) λmax 252, 335 nm (ε
8900, 13 900), λmin 228, 292 nm (ε 7300, 4200); 1H NMR (DMSO-d6) δ 12.81 (br s, 1H,
87
NH), 8.79 (s, 1H, H6), 8.10-8.24 (br s, 2H, Bz), 7.62 (t, J = 7.3 Hz, 1H, Bz), 7.53 (t, J =
7.6 Hz, 2H, Bz), 5.70 (d, J = 3.6 Hz, 1H, H1'), 5.57 (d, J = 3.9 Hz, 1H, 2'OH), 5.41 (t, J =
4.6 Hz , 1H, 5'OH), 5.10 (d, J = 5.9 Hz, 1H, 3'OH), 4.07-4.13 (m, 1H, H2'), 4.04 ("q", J =
5.9 Hz, 1H, H3'), 3.90-3.96 (m, 1H, H4'), 3.74-3.82 (m, 1H, H5'), 3.63 (ddd, J = 2.1, 4.4,
12.2 Hz, 1H, H5''); 13C NMR (DMSO-d6) δ 177.8 (Bz), 154.5 (C2), 147.2 (C4 ), 142.1
(C6), 136.1 (Bz), 132.8, 129.4, 128.4, 95.0 (C5), 89.7 (C1'), 84.5 (C4'), 74.2 (C2'), 68.6
(C3'), 59.5 (C5'); MS (ESI) m/z 426 (100, [79Br], MH+), 428 (98, [81Br], MH+). Anal.
Calcd for C16H16BrN3O6 • 0.5 MeOH (442.24): C, 44.81; H, 4.10; N, 9.50. Found: C,
44.62; H, 3.71; N, 9.13.
5'-O-(Tert-butyldimethylsilyl)-2',3'-O-isopropylideneuridine (49b)210. UV (MeOH)
λmax 276 nm, λmin 241 nm; 1H NMR (CDCl3) δ 9.23 (br s, 1H, NH), 7.91 (s, 1H, H6), 5.91
(d, J = 3.1 Hz, 1H, H1'), 4.73 (dd, J = 2.3, 6.2 Hz, 1H, H2'), 4.68 (dd, J = 3.1, 6.1 Hz, 1H,
H3'), 4.39 (“q”, J = 2.4 Hz, 1H, H4'), 3.93 (dd, J = 2.1, 11.7 Hz, 1H, H5'), 3.80 (dd, J =
2.8, 11.7 Hz, 1H, H5''), 1.58 (s, 3H, CH3), 1.35 (s, 3H, CH3), 0.90 (s, 9H, 3 x CH3), 0.11
(s, 6H, 2 x CH3).
8-Bromoadenosine (50b)211. UV (MeOH) λmax 264 nm, λmin 232 nm; 1H NMR (DMSO-
d6) δ 8.12 (s, 1H, H2), 7.58 (br s, 2H, NH2), 5.83 (d, J = 6.8 Hz, 1H, H1'), 5.52 (dd, J =
3.9, 8.6 Hz, 1H, 5'OH), 5.48 (d, J = 6.3 Hz, 1H, 2'OH), 5.25 (d, J = 4.4 Hz , 1H, 3'OH),
5.09 (q, J = 6.1 Hz, 1H, H2'), 4.20-4.17 (m, 1H, H3'), 3.99-3.96 (m, 1H, H4'), 3.68 (dt, J
= 3.9, 12.1 Hz, 1H, H5'), 3.55-3.49 (m, 1H, H5'').
8-Bromo-2'-deoxyadenosine (51b)54. 1H NMR (DMSO-d6) δ 8.11 (s, 1H, H2), 7.51 (br
s, 2H, NH2), 6.29 (“dd”, J = 6.7, 7.9 Hz, 1H, H1'), 5.34 (d, J = 4.2 Hz , 1H, 3'OH), 5.29
(“dd”, J = 4.4, 7.7 Hz, 1H, 5'OH), 4.50-4.46 (m, 1H, H3'), 3.90-3.87 (m, 1H, H4'), 3.65
88
(“dt”, J = 4.5, 11.9 Hz, 1H, H5'), 3.51-3.45 (m, 1H, H5''), 3.28-3.21 (m, 1H, H2'), 2.19
(ddd, J = 2.7, 6.5, 13.2 Hz, 1H, H2'').
8-Bromo-2',3',5'-tri-O-acetylguanosine (52b)212. UV (MeOH) λmax 263 nm, λmin 220
nm; 1H NMR (DMSO-d6) δ 10.95 (br s, 1H, NH), 6.63 (br s, 2H, NH2), 6.01 (“dd”, J =
4.5, 6.3 Hz, 1H, H1'), 5.88 (“d”, J = 4.4 Hz, 1H, H2'), 5.65 (t, J = 6.3 Hz , 1H, H3'), 4.42
(dd, J = 3.7, 11.9 Hz, 1H, H5'), 4.34-4.30 (m, 1H, H4'), 4.21 (dd, J = 6.3, 11.9 Hz, 1H,
H5''), 2.11 (s, 3H, Ac), 2.07 (s, 3H, Ac), 1.99 (s, 3H, Ac).
8-Bromoguanosine (53b)213. UV (MeOH) λmax 261 nm, λmin 223 nm; 1H NMR (DMSO-
d6) δ 10.87 (br s, 1H, NH), 6.51 (br s, 2H, NH2), 5.68 (“d”, J = 6.3 Hz, 1H, H1'), 5.44
(“d”, J = 6.2 Hz, 1H, H2'), 5.09 (d, J = 5.0 Hz , 1H, 2'OH), 5.01 (q, J = 6.0 Hz, 1H,
5'OH), 4.95 (t, J = 5.9 Hz, 1H, 3'OH), 4.15-4.12 (m, 1H, H3'), 3.87-3.84 (m, 1H, H4'),
3.68-3.62 (m, 1H, H5'), 3.54-3.48 (m, 1H, H5'').
8-Bromo-2'-deoxyguanosine (54b).214,215 UV (MeOH) λmax 261 nm, λmin 221 nm; 1H
NMR (DMSO-d6) δ 10.80 (br s, 1H, NH), 6.49 (br s, 2H, NH2), 6.15 (“t”, J = 7.3 Hz, 1H,
H1'), 5.25 (d, J = 4.3 Hz, 1H, 3'OH), 4.86 (t, J = 5.9 Hz, 1H, 5'OH), 4.41-4.37 (m, 1H,
H3'), 3.81-3.78 (m, 1H, H4'), 3.65-3.59 (m, 1H, H5'), 3.52-3.46 (m, 1H, H5''), 3.19-3.12
(m, 1H, H2'), 2.13-2.07 (m, 1H, H2'').
8-bromotoyocamycin (55).
Method A. NBS (195.8 mg, 1.1 mmol) was added to a stirred solution of 19c (291.3 mg,
1 mmol) in anhydrous DMF (8 mL) in a flame dried flask and the resulting pale-yellow
solution was stirred at room temperature for 2.5 h, by which time TLC showed >90%
conversion to a less polar product. Volatiles were removed from the clear, brown solution
using high vacuum rotary evaporator on a hot water bath (<80 oC). The resulting brown
89
gummy solid was purified using column chromatography (10%, MeOH/EtOAc) and
recrystallizaed from MeOH to give 55 as a colorless solid (166.6 mg, 45%): mp: 200-202
ºC; UV (MeOH): λmax 286 nm (ε 15 000), λmin 250 nm (ε 2200); 1H NMR (DMSO-d6): δ
8.20 (s, 1H, H2), 7.09 (br s, 2H, NH2), 5.94 (d, J = 6.6 Hz, 1H, H1'), 5.46 (d, J = 6.2 Hz,
1H, 2'-OH), 5.30-5.33 ("q", J = 4.1 Hz, 1H, 5'-OH), 5.25 (d, J = 4.8 Hz, 1H, 3'-OH), 5.08
(q, J = 6.0 Hz, 1H, H2'), 4.21 (m, 1H, H3'), 3.96 (q, J =3.8 Hz, 1H, H4'), 3.69 (dt, J
=4.20, 12.0 Hz, 1H, H5'), 3.50-3.57 (m, 1H, H5''); 13C NMR: δ= 156.1, 153.2, 149.9,
121.7, 114.1 (CN), 102.1, 91.0 (C1'), 87.4, 86.5 (C4'), 71.1 (C2'), 70.6 (C3'), 62.0 (C5').
Rf (EtOAc:iPrOH:H2O, 4:1:2) 0.69 (Rf 19c 0.68); HRMS (ESI): m/z [M+H]+ calcd for
C12H13BrN5O4+: 370.0145 and 372.0125; found 370.0093 and 371.9937.
Method B. DBH (552 mg, 1.93 mmol) was added to a suspension of 19c (750 mg, 2.57
mmol) in MeOH (120 mL) in a flame dried flask and the resulting yellow suspension was
stirred at room temperature for 5 min, by which time the solution became >80% clear.
After 15 min TLC showed >95% conversion to a less polar product and product began to
solidify out of the solution. After 30 min the colorless solid was collected using vacuum
filtration (710 mg). Volatiles in the mother liquor were removed using rotary evaporator
and the resulting colorless solid was recrystallized from MeOH (104 mg) to give 55 (814
mg, 85%): characterization data were in agreement with those reported in method 1.
8-bromosangivamycin (56).
Method A. NBS (267.1 mg, 1.5 mmol) was added to a stirred solution of 19b (309.2 mg,
1 mmol) in anhydrous DMF (8 mL) in a flame dried flask and the resulting pale-yellow
solution was stirred at room temperature for 1 h, by which time TLC showed >90%
conversion to a less polar product. Volatiles were removed from the clear, brown solution
90
using high vacuum rotary evaporator on a hot water bath (<80 oC). The resulting brown
gummy oil was column chromatographed (15%, MeOH/EtOAc) to give 56 as an off-
white solid (244.5 mg, 63%): mp: 174-175 ºC; UV (MeOH): λmax 286 nm (ε 14 100), λmin
256 nm (ε 5500); 1H NMR (DMSO-d6): δ 8.09 (s, 1H, H2), 8.00 (br s, 1H, NH2), 7.76 (br
s, 1H, NH2), 5.99 (d, J = 6.6 Hz, 1H, H1'), 5.55 (dd, J = 4.0 Hz, 1H, 5'-OH), 5.37 (d, J =
6.3 Hz, 1H, 2'-OH), 5.18 (d, J = 4.6 Hz, 1H, 3'-OH), 5.13 (q, J = 6.1 Hz, 1H, H2'), 4.20
("q", J = 4.2 Hz, 1H, H3'), 3.95 (q, J = 3.5 Hz, 1H, H4'), 3.68 (dt, J = 3.4, 12.0 Hz, 1H,
H5'), 3.49-3.55 (m, 1H, H5''); 13C NMR: δ = 165.8 (C=O), 157.1, 152.2, 149.6, 112.5,
112.2, 90.5 (C1'), 86.2 (C4'), 71.1 (C2'), 70.8 (C3'), 62.23 (C5'); Rf (EtOAc:iPrOH:H2O,
4:1:2) 0.45 (Rf 19b 0.35); HRMS (ESI): m/z [M+H]+ calcd for C12H15BrN5O5
+: 388.0251
and 390.0231; found 388.0265 and 390.0229.
Method B. DBH (101.6 mg, 1.1 mmol) was added to a clear, colorless solution of 19b
(100 mg, 0.0323 mmol) in MeOH (25 mL) in a flame dried flask and the resulting yellow
solution was stirred at room temperature for 30 min, by which time TLC showed >90%
conversion to a less polar product. Volatiles were removed from the clear, yellow
solution using rotary evaporator. The resulting off-white solid was purified using column
chromatography (10%, MeOH/EtOAc) to give 56 as a colorless solid (49.2 mg, 39%):
characterization data were in agreement with those reported in method 1.
Ethyl 2-(phenylthio)octanoate (72). Thiophenol (260 μL, 280 mg, 2.54 mmol) was
added to a stirred solution of NaH (60%, dispersion in paraffin liquid; 100.4 mg, 4.18
mmol) in anhydrous DMF (4 mL) at 0 oC. The resulting suspension was stirred at 0 oC for
30 minutes and at ambient temperature for 30 minutes, by which time bubbles (H2 gas)
ceased. The reaction flask was chilled again and Ethyl 2-bromooctanoate 71b (540 μL,
91
630 mg, 2.51 mmol) was added at 0 oC. The resultant clear, colorless solution was stirred
at 0 oC for 20 min and at ambient temperature for 2 hours, by which time TLC showed
exclusive conversion to a slightly more polar spot. Volatiles were evaporated and co-
evaporated with toluene (1 x) (vacuum pump) and the resulting pale gum was partitioned
between CHCl3 (20 mL) and NH4Cl/H2O (20 mL). The aqueous layer was extracted with
CHCl3 (2 x 5 mL) and the combined organic phase was washed with NaHCO3/H2O (25
mL), brine (25 mL), and dried (MgSO4). Volatiles were evaporated in vacuo and the
residue was column chromatographed (10% EtOAc in hexanes) to give 72216 (633 mg,
90%) as a pale oil: 1H NMR (CDCl3) δ 7.49-7.46 (m, 2H, Ph), 7.35-7.25 (m, 3H, Ph),
4.16-4.09 (m, 2H, CH2), 3.66 (dd, J = 6.6, 8.4 Hz, 1H, H2), 1.96-1.87 (m, 1H, H3), 1.82-
1.73 (m, 1H, H3'), 1.51-1.37 (m, 2H, H4, H4'), 1.36-1.26 (m, 6H, H5, H5', H6, H6', H7,
H7'), 1.19 (t, J = 7.1 Hz, 3H, CH3), 0.90 (“t”, J = 6.8 Hz, 3H, CH3); 13C NMR (CDCl3) δ
172.4, 133.8, 132.7, 128.9, 127.7, 61.0, 50.9, 31.7, 31.5, 28.8, 27.2, 22.5, 14.1, 14.0.
Ethyl 2-((4-chlorophenyl)thio)hexanoate (73). Treatment of ethyl 2-bromohexanoate
71a (500 μL, 610.5 mg, 2.74 mmol) with 4-chlorothiophenol/NaH/DMF as described for
the preparation of 72 gave 73217 as a pale yellow oil (763.6 mg, 97%): 1H NMR (CDCl3)
δ 7.41-7.37 (m, 2H, Ph), 7.30-7.26 (m, 2H, Ph), 4.17-4.09 (m, 2H, CH2), 3.61 (dd, J =
6.6, 8.4 Hz, 1H, H2), 1.94-1.84 (m, 1H, H3), 1.81-1.69 (m, 1H, H3'), 1.51-1.27 (m, 4H,
H4, H4', H5, H5'), 1.19 (t, J = 7.1 Hz, 3H, CH3), 0.91 (“t”, J = 7.1 Hz, 3H, CH3); 13C
NMR (CDCl3) δ 172.1, 134.11, 134.10, 132.1, 129.0, 61.1, 51.0, 31.3, 29.4, 22.2, 14.1,
13.8.
Ethyl 2-((4-chlorophenyl)thio)octanoate (74). Treatment of ethyl 2-bromooctanoate
71b (540 μL, 630 mg, 2.51 mmol) with 4-chlorothiophenol/NaH/DMF as described for
92
the preparation of 72 gave 74 as a colorless oil (664 mg, 84%): 1H NMR (CDCl3) δ 7.39-
7.37 (m, 2H, Ph), 7.27-7.24 (m, 2H, Ph), 4.15-4.07 (m, 2H, CH2), 3.60 (dd, J = 6.7, 8.3
Hz, 1H, H2), 1.92-1.81 (m, 1H, H3), 1.77-1.68 (m, 1H, H3'), 1.48-1.35 (m, 2H, H4, H4'),
1.33-1.27 (m, 6H, H5, H5', H6, H6', H7, H7'), 1.18 (t, J = 7.1 Hz, 3H, CH3), 0.87 (“t”, J =
6.8 Hz, 3H, H8, H8', H8''); 13C NMR (CDCl3) δ 172.0, 134.1, 134.0, 132.2, 129.0, 61.0,
51.0, 31.6, 31.5, 28.8, 27.2, 22.5, 14.1, 14.0.
Ethyl 2-((4-methoxyphenyl)thio)hexanoate (75). Treatment of ethyl 2-bromohexanoate
71a (500 μL, 610.5 mg, 2.74 mmol) with 4-methoxythiophenol/NaH/DMF as described
for the preparation of 72 gave 75218 as a colorless oil (739.3 mg, 96%): 1H NMR (CDCl3)
δ 7.36-7.31 (m, 2H, Ph), 6.78-6.74 (m, 2H, Ph), 4.02 (“q”, J = 7.2 Hz, 2H, CH2), 3.41
(dd, J = 6.6, 8.5 Hz, 1H, H2), 1.82-1.75 (m, 1H, H3), 1.68-1.59 (m, 1H, H3'), 1.42-1.32
(m, 1H, H4), 1.30-1.21 (m, 3H, H4', H5, H5'), 1.10 (t, J = 7.1 Hz, 3H, CH3), 0.82 (t, J =
7.1 Hz, 3H, CH3); 13C NMR (CDCl3) δ 172.2, 160.1, 136.2, 123.4, 114.4, 60.7, 55.2,
51.6, 31.1, 29.3, 22.3, 14.1, 13.8.
Typical procedure for preparation of ethyl difluoroalkanoate (76 and 77). DBH was
added to a stirred solution of ester 72 or 73 (0.5 mmol) and Py.9HF (3 mmol) in CH2Cl2
(2 mL) at ambient temperature. The resulting brown solution was stirred at ambient
temperature or at 35 oC for the respective times as shown in Table 1. The reaction flask
was cooled to room temperature, quenched by addition of ice-cold water, diluted with
CH2Cl2, and neutralized with drop-wise addition of conc. NH4OH. Organic layer was
separated and aqueous layer was back extracted (2 x CH2Cl2). Combined organic layer
was washed with 1N HCl, brine, dried (MgSO4) and concentrated in vacuo to give crude
difluorinated product as a brown oil, with data as reported.219 76219 had: 1H NMR
93
(CDCl3) δ 4.25 (q, 3JH,H = 7.2 Hz, 2H, CH2), 2.09-1.88 (m, 2H, H3, H3'), 1.42-1.30 (m,
4H, H4, H4', H5, H5'), 1.28 (t, 3JH,H = 7.1 Hz, 3H, CH3), 0.85 (t, 3JH,H = 7.2 Hz, 3H, H6,
H6', H6''); 13C NMR (CDCl3) δ 164.4 (t, 2JF,C = 33.2 Hz, C1), 116.4 (t, 1JF,C = 249.8 Hz,
C2), 63.0 (CH2), 34.5 (t, 2JF,C = 23.2 Hz, C3), 23.5 (t, 3JF,C = 4.3 Hz, C4), 22.2 (C5), 13.9
(CH3), 13.7 (C6); 19F NMR (CDCl3) δ –105.92 ppm (t, 3JF-H = 16.8 Hz); GC-MS (tR = 7.5
min) 151 (l.1), 124 (11.3), 116 (18.2), 87 (44.9), 67 (20.9), 55 (3.6);
77219 had: 1H NMR (CDCl3) δ 4.25 (q, 3JH,H = 7.2 Hz, 2H, CH2), 2.03-1.91 (m, 2H), 1.45-
1.18 (m, 11H), 0.81 (“t”, 3JH,H = 6.8 Hz, 3H, H6, H6', H6''); 13C NMR (CDCl3) δ 164.5 (t,
2JF,C = 33.0 Hz, C1), 116.4 (t, 1JF,C = 249.7 Hz, C2), 62.7 (CH2), 34.5 (t, 2JF,C = 23.2 Hz,
C3), 31.4, 28.7, 22.4, 21.4 (t, 3JF,C = 4.3 Hz, C4), 13.9; 19F NMR (CDCl3) δ –105.89 ppm
(t, 3JF-H = 17.0 Hz).
5-O-Benzyl-2,3-dideoxy-2-fluoro-2-(phenylsulfanyl)ribose (84) and 5-O-Benzyl-2,3-
dideoxy-2-fluoro-2-(phenylsulfinyl)ribose (85). 83 (31 mg, 0.1 mmol) was treated with
Py.9HF (185 μL, 0.8 mmol) and DBH (114 mg, 0.4 mmol) in anhydrous CH2Cl2 (3 mL)
according to general desulfurization-difluorination procedure gave 40 mg of yellow oil
after aqueous workup: 19F-NMR (CDCl3) δ -150.86 (“t”, 1F, J = 26.4 Hz), -149.33 (dd,
0.4F, J = 20.3, 27.3 Hz), -139.24 to -138.93 (m, 0.6 F); HRMS (ESI) m/z 355.2857
[M+Na]+, calcd for C18H17FNaO3S+ 355.0775; m/z 371.2611 [M+Na]+, calcd for
C18H17FNaO4S+ 371.0724.
2'-S-(4-Chlorophenyl)-2'-thiouridine (87). Slight modification of published
procedure.133 PPh3 (464.3 mg, 1.77 mmol) was added to a stirred solution of 4-
chlorothiophenol (1.28 g, 8.84 mmol) in anhydrous DMF (10 mL) and the resulting
solution was stirred at ambient temperature for 30 minutes. 2,2'-anhydro-1-β-D-
94
arabinofuranosyluracil220 (1 g, 4.42 mmol) was added to the reaction mixture and the
resulting solution was refluxed (153 oC) for 18 h. Volatiles were evaporated, co-
evaporated with toluene (2 x). The resultant brown gum was triturated with hexanes (3 x)
and the liquid was pipetted out. The remaining brownish gummy solid was triturated with
ether and the resultant pale green solid was vacuum filtered, washed with minimal
amount of ice-cold methanol to give 2'-S-(4-Chlorophenyl)-2'-thiouridine (87; 1.1 g, 67
%) as a colorless solid: 1H NMR (DMSO-d6)133 δ 11.14 (br s, 1H, NH), 7.61 (d, J = 8.1
Hz, 1H, H6), 7.35 (d, J = 8.5 Hz, 2H, Ph), 7.28 (d, J = 8.5 Hz, 2H, Ph), 6.33 (d, J = 9.0
Hz, 1H, H1'), 5.91 (d, J = 5.6 Hz, 1H, 3'OH), 5.50 (d, J = 8.1 Hz, 1H, H5), 5.11 (t, J = 5.0
Hz, 1H, 5'OH), 4.34 (t, J = 5.2 Hz, 1H, H3'), 3.94-3.92 (m, 1H, H4'), 3.89 (dd, J = 5.3,
9.0 Hz, 1H, H2'), 3.59 (“t”, J = 4.4 Hz, 2H, H5', H5''); 13C NMR (DMSO-d6) δ 162.5
(C4), 150.4 (C2), 139.8 (C6), 133.1 (Ph), 132.3 (Ph), 132.0 (Ph), 128.8 (Ph), 102.3 (C5),
87.8 (C1'), 86.7 (C4'), 72.1 (C3'), 61.4 (C5'), 54.5 (C2').
3',5'-di-O-Acetyl-2'-S-(4-chlorophenyl)-2'-thiouridine (89a). DMAP (18.3 mg, 0.15
mmol) was added to a stirred suspension of 87 (1.1 g, 2.97 mmol) in Ac2O (842 μL, 909
mg, 8.91 mmol) and the resulting suspension was stirred at ambient temperature
overnight (15 h). MeOH was added, the reaction mixture was stirred at ambient
temperature for 30 min, and volatiles were evaporated (vacuum pump, <25 oC). MeOH
was added and evaporated, and the resulting gum was partitioned between CHCl3 (50
mL) and H2O (50 mL). The aqueous layer was extracted with CHCl3, and the combined
organic phase was washed with NaHCO3/H2O, 1N HCl, brine, and dried (MgSO4).
Volatiles were evaporated in vacuo and the residue was dried on vacuum pump to give
3',5'-di-O-Acetyl-2'-S-(4-chlorophenyl)-2'-thiouridine (89a; 1.34 g, 99%) as a colorless
95
foam, with sufficient purity to proceed to next step: 1H NMR (CDCl3) δ 10.12 (br s, 1H,
NH), 7.27 (d, J = 8.3 Hz, 2H, Ph), 7.22 (d, J = 8.2 Hz, 1H, H6), 7.14 (d, J = 8.4 Hz, 2H,
Ph), 6.14 (d, J = 8.5 Hz, 1H, H1'), 5.63 (d, J = 8.2 Hz, 1H, H5), 5.38 (“d”, J = 6.0 Hz,
1H, H3'), 4.30-4.19 (m, 3H, H4', H5', H5''), 3.81 (“dd”, J = 6.3, 8.2 Hz, 1H, H2'), 2.07 (s,
3H, CH3), 2.00 (s, 3H, CH3); 13C NMR (CDCl3) δ 170.3, 169.9 (2 x C=O), 163.2 (C4),
150.4 (C2), 139.2 (C6), 134.6 (Ph), 134.2 (Ph), 130.6 (Ph), 129.4 (Ph), 103.4 (C5), 89.0
(C1'), 81.0 (C4'), 73.7 (C3'), 63.5 (C5'), 53.5 (C2'), 20.7, 20.6 (2 x s, 2 x CH3).
N3-Benzyl-3',5'-di-O-benzyl-2'-S-(4-chlorophenyl)-2'-thiouridine (89b). NaH (60%,
dispersion in paraffin liquid; 216 mg, 9 mmol) was added to a stirred solution of 87
(556.2 mg, 1.5 mmol) in anhydrous DMF (6 mL) at 0 oC. The resulting suspension was
stirred at 0 oC for 30 minutes and at ambient temperature for 30 minutes, by which time
bubbles (H2 gas) ceased. The reaction flask was chilled again and benzyl bromide (0.93
mL, 1.3 g, 7.8 mmol) was added at 0 oC. Bubbles were observed for a few minutes once
the ice-bath was removed and the resultant pale solution was stirred at ambient
temperature for 4 hours, by which time TLC showed a major (90%) less polar spot.
Volatiles were evaporated and co-evaporated with toluene (1 x) (vacuum pump) and the
resulting syrup was partitioned between CHCl3 (20 mL) and NH4Cl/H2O (20 mL). The
aqueous layer was extracted with CHCl3 (2 x 5 mL) and the combined organic phase was
washed with NaHCO3/H2O (25 mL), brine (25 mL), and dried (MgSO4). Volatiles were
evaporated in vacuo and the residue was column chromatographed (20% EtOAc in
hexanes) to give 89b (827 mg, 86%) as a colorless foam: 1H NMR (CDCl3) δ 7.53-7.51
(m, 2H, Ph), 7.43-7.27 (m, 14H, Ph, H6), 7.15-7.11 (m, 2H, Ph), 6.93-6.90 (m, 2H, Ph),
6.47 (d, J = 8.3 Hz, 1H, H1'), 5.33 (d, J = 8.1 Hz, 1H, H5), 5.05-4.97 (m, 2H, benzylic),
96
4.71-4.64 (m, 2H, benzylic), 4.56 (“d”, J = 11.0 Hz, 1H, benzylic), 4.50 (“d”, J = 11.0
Hz, 1H, benzylic), 4.33-4.30 (m, 2H, H3', H4'), 3.81-3.77 (m, 2H, H2', H5'), 3.60 (dd, J =
1.7, 10.4 Hz, 1H, H5''); 13C NMR (CDCl3) δ 162.0, 150.9, 137.4, 137.2, 137.1, 136.8,
133.9, 133.8, 131.7, 129.5, 129.1, 128.8, 128.6, 128.4, 128.1, 128.0, 127.8, 127.7, 102.3
(C5), 90.2 (C1'), 82.3 (C4'), 80.3 (C3'), 73.9 (CH2), 72.4 (CH2), 70.6 (C5'), 55.7 (C2'),
44.1 (CH2).
N3-Benzyl-3',5'-di-O-benzyl-2'-S-(4-methoxyphenyl)-2'-thiouridine (90b). Treatment
of 88134 (1.47 g, 4.0 mmol) with NaH (576 mg, 24 mmol) and BnBr (2.5 mL, 3.6 g, 20.8
mmol) as described for 87 → 89b gave 90b as a colorless foam (2.2 g, 86%): 1H NMR
(CDCl3) δ 7.58 (“d”, J = 6.9 Hz, 2H, Ph), 7.40-7.23 (m, 13H, Ph), 7.14-7.11 (m, 3H, Ph,
H6), 6.56 (d, J = 8.8 Hz, 1H, H1'), 6.33 (d, J = 8.7 Hz, 2H, Ph), 5.11 (d, J = 8.1 Hz, 1H,
H5), 5.04 (“d”, J = 13.5 Hz, 1H, benzylic), 4.95 (“d”, J = 13.5 Hz, 1H, benzylic), 4.64 (s,
2H, benzylic), 4.49 (“d”, J = 11.0 Hz, 1H, benzylic), 4.39 (“d”, J = 11.0 Hz, 1H,
benzylic), 4.29 (d, J = 5.3 Hz, 1H, H4'), 4.25 (br s, H3'), 3.70-3.65 (m, 2H, H2', H5'),
3.60 (s, 3H, OCH3), 3.54-3.51 (“m”, 1H, H5''); 13C NMR (CDCl3) δ 170.8, 162.0, 159.8,
151.1, 137.4, 136.9, 135.3, 129.8, 128.6, 128.5, 128.3, 128.2, 127.9, 127.8, 127.8, 127.7,
123.3, 114.6, 102.0 (C5), 90.4 (C1'), 82.0 (C3'), 80.9 (C4'), 73.8 (CH2), 72.2 (CH2), 70.8
(C5'), 56.3 (C2'), 55.4 (OCH3), 44.1 (CH2).
N3-Benzyl-3',5'-di-O-benzyl-2'-deoxy-2'-[(4-methoxyphenyl)sulfinyl]uridine [93
(R/S-S)]. MCPBA (750.7 mg of 70% reagent, 4.35 mmol) in CH2C12 (70 mL) was added
drop-wise to a stirred solution of 90b (2.2 g, 3.45 mmol) in CH2C12 (35 mL) at -50 oC,
and the temperature was allowed to rise to -30 oC in ~20 minutes. TLC showed > 80%
conversion to a more polar product. The solution was poured into NaHCO3/H2O (10 mL).
97
The aqueous layer was extracted with CHCl3 (2 x 5 mL) and the combined organic phase
was washed with H2O (20 mL), brine (25 mL), and dried (MgSO4). Volatiles were
evaporated in vacuo and the residue was column chromatographed (25% EtOAc in
hexanes) to give 93 (2' R/S-S, ~1:1) (1.8 g, 80%) as a colorless foam: 1H NMR (CDCl3) δ
7.51-7.28 (m, 17.5H, Ph, H6), 7.10 (d, J = 7.8 Hz, 0.5H, H6), 7.03 (d, J = 8.0 Hz, 0.5H,
H1'), 6.83 (d, J = 8.0 Hz, 0.5H, H1'), 6.49 (d, J = 9.6 Hz, 1H, Ph), 6.44 (d, J = 8.1 Hz,
1H, Ph), 5.36 (d, J = 7.7 Hz, 0.5H, H5), 5.04 (d, J = 8.1 Hz, 0.5H, H5), 4.96-4.26 (m, 8H,
benzylic, H2', H3'), 3.86-3.43 (m, 6H, CH3, H4', H5', H5'');
13C NMR (CDCl3) for major isomer (extracted data): δ 162.9, 161.6, 150.6, 137.11,
137.10, 137.0, 136.5, 132.5, 129.8, 128.8, 128.6, 128.44, 128.38, 128.2, 127.9, 127.2,
114.6, 102.5 (C5), 82.9 (C1'), 82.1 (C4'), 80.9 (C3'), 74.0 (CH2), 73.4 (CH2), 72.7 (C5'),
70.9 (C2'), 55.6 (OCH3), 44.1 (CH2).
3',5'-di-O-Acetyl-2'-deoxy-2'-[(4-chlorophenyl)sulfonyl]uridine (91a). MCPBA (70%
reagent, 250 mg, 1.45 mmol) was added to a stirred solution of 89a (227.4 mg, 0.5 mmol)
in CH2Cl2 (15 mL) and the resultant colorless solution was stirred at ambient temperature
for 12h. TLC showed complete conversion to a more polar product. The reaction mixture
was poured into NaHCO3/H2O (10 mL). The aqueous layer was extracted with CHCl3 (2
x 5 mL) and the combined organic phase was washed with H2O (20 mL), brine (25 mL),
and dried (MgSO4). Volatiles were evaporated in vacuo to give 91a (241 mg, 99%) as a
colorless foam. Data for the crude sample: 1H NMR (CDCl3) δ 9.51 (br s, 1H, NH), 7.86
(d, J = 8.6 Hz, 2H, Ph), 7.55 (d, J = 8.6 Hz, 2H, Ph), 7.17 (d, J = 8.2 Hz, 1H, H6), 6.15
(d, J = 6.2 Hz, 1H, H1'), 5.72-5.66 (m, 2H, H5, H3'), 4.6 (t, J = 6.7 Hz, 1H, H2'), 4.42 (q,
J = 4.6 Hz, 1H, H4'), 4.36 (dd, J = 3.7, 12.5 Hz, 1H, H5'), 4.25 (dd, J = 4.9, 12.2 Hz, 1H,
98
H5''), 2.09 (s, 3H, CH3), 2.07 (s, 3H, CH3); 13C NMR (CDCl3) δ 170.4, 169.9 (2 x C=O),
162.9 (C4), 149.6 (C2), 141.7 (C6), 141.2 (Ph), 136.9 (Ph), 130.2 (Ph), 130.1 (Ph), 103.5
(C5), 87.7 (C1'), 80.9 (C4'), 71.2 (C3'), 65.1 (C5'), 62.4 (C2'), 20.71, 20.70 (2 x CH3).
N3-Benzyl-3',5'-di-O-benzyl-2'-deoxy-2'-[(4-chlorophenyl)sulfonyl]uridine (91b).
MCPBA (250.2 mg of 70% reagent, 1.45 mmol) in CH2C12 (6 mL) was added drop-wise
to a stirred solution of 89b (320.6 mg, 0.50 mmol) in CH2C12 (4 mL) and the resulting
clear solution was stirred at ambient temperature overnight. Saturated NaHCO3/H2O (10
mL) was added, stirring was continued for 10 min, and the organic layer was separated.
The aqueous layer was extracted with CHCl3 (2 x 5 mL) and the combined organic phase
was washed with H2O (20 mL), brine (25 mL), and dried (MgSO4). Volatiles were
evaporated in vacuo and the residue was column chromatographed (20% EtOAc in
hexanes) to give 91b (269.3 mg, 80%) as a colorless foam: 1H NMR (CDCl3) δ 7.59-7.56
(m, 2H, Ph), 7.55-7.51 (m, 2H, Ph), 7.48-7.28 (m, 14H, Ph, H6), 6.98-6.94 (m, 2H, Ph),
6.66 (d, J = 8.6 Hz, 1H, H1'), 5.37 (d, J = 8.2 Hz, 1H, H5), 5.08 (“d”, J = 13.7 Hz, 1H,
benzylic), 5.02 (“d”, J = 13.7 Hz, 1H, benzylic), 4.68 (“d”, J = 11.5 Hz, 1H, benzylic),
4.63 (dd, J = 1.8, 5.4 Hz, 1H, H3'), 4.58 (“d”, J = 11.5 Hz, 1H, benzylic), 4.53 (“d”, J =
10.9 Hz, 1H, benzylic), 4.45 (“d”, J = 11.0 Hz, 1H, benzylic), 4.24 (“d”, J = 1.9 Hz, 1H,
H4'), 4.09 (dd, J = 5.4, 8.6 Hz, 1H, H2'), 3.70 (dd, J = 2.3, 10.6 Hz, 1H, H5'), 3.46 (dd, J
= 2.0, 10.6 Hz, 1H, H5''); 13C NMR (CDCl3) δ 161.8, 150.7, 140.7, 137.4, 137.1 (C6),
136.8, 136.6, 136.4, 130.2, 129.5, 129.1, 128.8, 128.6, 128.5, 128.3, 128.23, 128.16,
127.8, 103.0 (C5), 84.8 (C1'), 82.5 (C4'), 79.7 (C3'), 74.0 (CH2), 73.1 (CH2), 70.1 (C5'),
68.9 (C2'), 44.3 (CH2).
N3-Benzyl-3',5'-di-O-benzyl-2'-deoxy-2'-[(4-methoxyphenyl)sulfonyl]uridine (92b).
99
Treatment of 90b (32 mg, 0.05 mmol) with MCPBA (26 mg of 70% reagent, 0.15 mmol)
in CH2Cl2 (3 mL) as described for 89b → 91b gave 92b (27 mg, 81%) as a colorless
foam: 1H NMR (CDCl3) δ 7.61-7.60 (m, 2H, Ph), 7.51-7.48 (m, 2H, Ph), 7.43-7.25 (m,
13H, Ph), 7.10 (d, J = 8.2 Hz, 1H, H6), 6.75 (d, J = 9.0 Hz, 1H, H1'), 6.39-6.35 (m, 2H,
Ph), 5.16 (d, J = 8.1 Hz, 1H, H5), 5.07 (“d”, J = 13.5 Hz, 1H, benzylic), 4.99 (“d”, J =
13.5 Hz, 1H, benzylic), 4.80 (“d”, J = 11.7 Hz, 1H, benzylic), 4.65-4.62 (m, 2H, benzylic,
H3'), 4.50 (“d”, J = 10.8 Hz, 1H, benzylic), 4.39 (“d”, J = 10.8 Hz, 1H, benzylic), 4.22
(“d”, J = 1.6 Hz, 1H, H4'), 4.05 (dd, J = 5.2, 9.0 Hz, 1H, H2'), 3.67-3.63 (m, 4H, OCH3,
H5'), 3.45 (dd, J = 2.0, 10.5 Hz, 1H, H5''); 13C NMR (CDCl3) δ 171.1, 163.9, 161.9,
150.7, 137.2 (C6), 137.0, 136.9, 136.6, 130.6, 130.4, 129.8, 128.8, 128.5, 128.4, 128.2,
128.1, 127.9, 114.2, 102.7 (C5), 85.0 (C1'), 82.6 (C4'), 80.0 (C3'), 73.9 (CH2), 73.1
(CH2), 70.4 (C5'), 68.4 (C2'), 55.8 (OCH3), 44.3 (CH2).
N3-Benzyl-3',5'-di-O-benzyl-2'-fluoro-2'-S-(4-methoxyphenyl)-2'-thiouridine (94b).
DAST (0.15 mL, 183 mg, 1.14 mmol) was added to a mixture of 93 (2'-R/S-S, ~1:1)
(326.4 mg, 0.5 mmol) and SbC13 (23 mg, 0.1 mmol) in CH2Cl2 (10 mL), and the
resulting pale solution was stirred at ambient temperature overnight (13 h). Cold
saturated NaHCO3/H2O (10 mL) was added carefully, stirring was continued for 30 min,
the organic layer was separated. The aqueous layer was extracted with CHCl3 (2 x 5 mL)
and the combined organic phase was washed with H2O (20 mL), brine (25 mL), dried
(MgSO4) and evaporated in vacuo to give yellow oil. Gradient flash chromatography (0
→ 30% EtOAc in hexanes) gave 94b as a mixture of diastereomers (2'-R/S-S, ~ 4:1) (pale
yellow foam 186.6 mg, 57%): 1H NMR (CDCl3) δ 7.55-7.26 (m, 18H, Ph, H6), 6.84 (“d”,
J = 8.7 Hz, 2H, Ph), 6.51 (d, J = 14.7 Hz, 0.8H, H1'), 6.40 (br s, 0.2H, H1'), 5.68 (d, J =
100
8.1 Hz, 0.8H, H5), 5.41 (d, J = 8.2 Hz, 0.2H, H5), 5.14-5.01 (m, 1H, benzylic), 4.93-4.87
(m, 1H, benzylic), 4.68-4.43 (m, 4H, benzylic), 4.23-4.18 (m, 2H, H3', H4'), 3.91-3.79
(m, 4H, CH3, H5'), 3.72-3.60 (m, 1H, H5''); 19F NMR (CDCl3) δ –132.53 ppm (br s,
0.8F), –131.16 ppm (br s, 0.2F).
13C NMR (CDCl3) for major isomer (extracted data): δ 162.3, 161.0, 150.2, 138.8, 137.6,
137.1, 136.6, 135.1, 135.0, 129.0, 128.59, 128.57, 128.4, 128.3, 128.1, 127.80, 127.6,
118.84, 118.82, 114.8, 109.1 (d, 1JC2'-F = 237.1 Hz, C2'), 102.0, 90.5 (d, 2JC1'-F = 47.9 Hz,
C1'), 80.1 (C4'), 79.6 (d, 2JC3'-F = 18.1 Hz, C3'), 73.5 (CH2), 73.1 (CH2), 67.5 (C5'), 55.4
(CH3), 44.1 (CH2).
N3-Benzyl-3',5'-di-O-benzyl-2'-deoxy-2'-fluoro-2'-[(4-
methoxyphenyl)sulfonyl]uridine (95b). MCPBA (241.6 mg of 70% reagent, 1.4 mmol)
in CH2C12 (5 mL) was added drop-wise to a stirred solution of 94 (327.4 mg, 0.50 mmol)
in CH2C12 (3 mL) and the resulting clear solution was stirred at ambient temperature
overnight. Saturated NaHCO3/H2O (10 mL) was added, stirring was continued for 10
min, and the organic layer was separated. The aqueous layer was extracted with CHCl3 (2
x 5 mL) and the combined organic phase was washed with H2O (20 mL), brine (25 mL),
and dried (MgSO4). Volatiles were evaporated in vacuo and the residue was column
chromatographed (20% EtOAc in hexanes) to give 95b as a mixture of diastereomers (2'-
R/S-S, ~ 3:1) (colorless foam, 298.7 mg, 87%): 1H NMR (CDCl3) δ 7.90 (d, J = 8.4 Hz,
0.5H, Ph), 7.78 (d, J = 8.3 Hz, 1.5H, Ph), 7.54 (d, J = 7.9 Hz, 1H, H6), 7.55-7.12 (m,
15H, Ph), 6.95 (“d”, J = 8.9 Hz, 1.5H, Ph), 6.89 (“d”, J = 8.5 Hz, 0.5H, Ph), 6.70 (br s,
0.25H, H1'), 6.30 (d, J = 18.6 Hz, 0.75H, H1'), 5.73 (d, J = 8.1 Hz, 0.75H, H5), 5.27 (br
s, 0.25H, H5), 5.06 (d, J = 13.7 Hz, 0.75H, benzylic), 5.00 (“d”, J = 13.7 Hz, 0.25H,
101
benzylic), 4.92-4.80 (m, 2H, benzylic, H3'), 4.58 (d, J = 11.6 Hz, 1H, benzylic), 4.49-
4.27 (m, 3H, benzylic), 4.11-4.06 (m, 1H, H4'), 3.90 (s, 2.25H, CH3), 3.86 (s, 0.75H,
CH3), 3.81-3.87 (m, 0.25H, H5'), 3.75 (dd, J = 2.3, 11.2 Hz, 0.75H, H5'), 3.54 (“dd”, J =
3.7, 11.1 Hz, 1H, H5''); 19F NMR (CDCl3) δ –158.89 ppm (“t”, J = 16.3 Hz, 0.75F), –
156.50 ppm (d, J = 19.5 Hz, 0.25F).
13C NMR (CDCl3) for major isomer: δ 165.0, 150.2, 139.3 (C6), 137.3, 136.8, 136.2,
131.38, 131.37, 129.1, 128.9, 128.6, 128.51, 128.50, 128.44, 128.36, 128.3, 128.1, 127.8,
127.7, 126.5, 114.5, 108.3 (d, 1JC2'-F = 237.0 Hz, C2'), 101.8 (C5), 87.8 (d, 2JC1'-F = 38.1
Hz, C1'), 79.5 (C4'), 75.5 (d, 2JC3'-F = 15.2 Hz, C3'), 73.8 (CH2), 73.5 (CH2), 67.1 (C5'),
55.9 (CH3), 44.1 (CH2).
5-Bromo-3',5'-di-O-acetyl-2'-S-(4-chlorophenyl)-2'-thiouridine (96a) and 5-Bromo-
3',5'-di-O-acetyl-2'-deoxy-2'-[(4-chlorophenyl)sulfinyl]uridine [97 (R/S-S)]. Py.9HF
(352 μL, 1.53 mmol) was added to a chilled solution of DBH (20.8 mg, 0.073 mmol) in
dry CH2Cl2 (1 mL) at -78 oC. The resulting pale solution was stirred at -78 oC for 10
minutes and a solution of substrate 89a (30mg, 0.066 mmol) in dry CH2Cl2 (2 mL) was
added via syringe. The resultant orange solution was stirred at -78 oC for 2h and was
brought to -30 oC over 45 minutes. The reaction mixture was diluted with CH2Cl2 (5 mL),
washed with NaHCO3/H2O (5 mL), H2O (5 mL), brine (5 mL), dried (MgSO4). Volatiles
were evaporated in vacuo and the yellow residue was column chromatographed (30% →
60% EtOAc in hexanes) to give 96a (14.4 mg, 41%) as a colorless oil. Also isolated were
97 (6.5 mg, 18%, faster moving isomer) as a colorless oil and 97 (10.9 mg, 30%, slower
moving isomer) as a colorless oil.
102
96a had: 1H NMR (CDCl3) δ 8.48 (br s, 1H, NH), 7.09-7.06 (m, 2H, Ph), 7.02-6.99 (m,
2H, Ph), 6.98 (s, 1H, H6), 5.94 (d, J = 8.1 Hz, 1H, H1'), 5.12 (dd, J = 2.6, 5.9 Hz, 1H,
H3'), 4.12 (dd, J = 4.0, 12.9 Hz, 1H, H5'), 4.06-4.02 (m, 2H, H4', H5''), 3.47 (dd, J = 5.9,
8.0 Hz, 1H, H2'), 1.893-1.891 (“m”, 6H, 2 x CH3); 13C NMR (CDCl3) δ 170.03 (C=O),
169.98 (C=O), 158.3, 149.5, 138.2 (C6), 135.2, 134.3 (Ph), 130.4, 129.7 (Ph), 98.0 (C5),
89.3 (C1'), 81.3 (C4'), 73.6 (C3'), 63.5 (C5'), 54.5 (C2'), 20.9 (CH3), 20.6 (CH3).
97 (faster moving isomer) had: 1H NMR (CDCl3) δ 8.38 (br s, 1H, NH), 7.56-7.50 (m,
4H, Ph), 7.10 (s, 1H, H6), 5.89 (d, J = 4.0 Hz, 1H, H1'), 5.51 (t, J = 8.2 Hz, 1H, H3'),
4.45 (dd, J = 2.8, 12.3 Hz, 1H, H5'), 4.40-4.36 (m, 1H, H4'), 4.23 (dd, J = 4.8, 12.3 Hz,
1H, H5''), 3.92 (dd, J = 4.2, 8.2 Hz, 1H, H2'), 2.29 (s, 3H, CH3), 2.12 (s, 3H, CH3); 13C
NMR (CDCl3) δ 170.6 (C=O), 170.4 (C=O), 157.9, 148.1, 140.6 (C6), 138.5, 138.4,
130.1 (Ph), 125.5 (Ph), 97.3 (C5), 85.3 (C1'), 80.3 (C4'), 72.1 (C3'), 67.3 (C2'), 62.1
(C5'), 20.8 (CH3), 20.6 (CH3).
97 (slower moving isomer) had: 1H NMR (CDCl3) δ 8.55 (br s, 1H, NH), 7.59-7.56 (m,
2H, Ph), 7.42-7.39 (m, 2H, Ph), 7.15 (s, 1H, H6), 6.13 (d, J = 8.4 Hz, 1H, H1'), 5.51 (dd,
J = 2.6, 6.2 Hz, 1H, H3'), 4.30 (dd, J = 3.2, 12.1 Hz, 1H, H5'), 4.25 (q, J = 2.9 Hz, 1H,
H4'), 4.17 (dd, J = 2.9, 12.1 Hz, 1H, H5''), 3.72 (dd, J = 6.2, 8.3 Hz, 1H, H2'), 2.13 (s,
3H, CH3), 2.11 (s, 3H, CH3); 13C NMR (CDCl3) δ 169.8 (C=O), 169.7 (C=O), 157.5,
148.7, 139.8 (C6), 139.0, 137.6, 130.1 (Ph), 127.1 (Ph), 98.2 (C5), 83.1 (C1'), 81.6 (C4'),
73.4 (C3'), 69.8 (C2'), 63.4 (C5'), 21.0 (CH3), 20.6 (CH3).
3',5'-di-O-Acetyl-5-iodo-2'-S-(4-chlorophenyl)-2'-thiouridine (96b). NIS (68 mg, 0.42
mmol) was added to a stirred solution of 89a (46 mg, 0.1 mmol) and DAST (80 μL, 0.6
mmol) in anhydrous CH2Cl2 (2 mL) at 0 oC and the resulting purple solution was stirred
103
at 0 oC for 6 h and at ambient temperature for 32 h (total reaction time: 36 h). The
reaction was quenched by addition of sat. Na2S2O3 (5 mL), diluted with CH2Cl2 (5 mL).
Organic layer was separated and aqueous layer was back extracted (2 x CH2Cl2).
Combined organic layer was washed with NaHCO3/H2O (10 mL), brine (10 mL), dried
(MgSO4), and filtered. Volatiles were evaporated in vacuo and the brown residue was
column chromatographed (25% → 50% EtOAc in hexanes) to give 96b (42.5 mg, 73%)
as a colorless oil.
Analogous treatment of 89a (46 mg, 0.1 mmol) with NIS (68 mg, 0.42 mmol) and
DAST (80 μL, 0.6 mmol) at 35 oC for 24 h gave 96b (35 mg, 60%) as a pale-yellow oil.
1H NMR (CDCl3) δ 9.07 (br s, 1H, NH), 7.60 (s, 1H, H6), 7.39-7.35 (m, 2H, Ph), 7.31-
7.28 (m, 2H, Ph), 6.23 (d, J = 8.4 Hz, 1H, H1'), 5.43 (dd, J = 2.2, 5.8 Hz, 1H, H3'), 4.44-
4.31 (m, 3H, H4', H5', H5''), 3.77 (dd, J = 5.9, 8.3 Hz, 1H, H2'), 2.21 (s, 3H, CH3), 2.19
(s, 3H, CH3); 13C NMR (CDCl3) δ 171.3, 170.0 (2 x C=O), 159.2 (C4), 149.8 (C2), 143.3
(C6), 135.2 (Ph), 134.3 (Ph), 130.4 (Ph), 129.8 (Ph), 89.0 (C1'), 81.3 (C4'), 73.7 (C3'),
69.7 (C5), 63.6 (C5'), 54.5 (C2'), 21.1, 20.7 (2 x s, 2 x CH3).
3-N-benzyl-3',5'-di-O-benzyl-5-bromo-2'-deoxy-2'-fluoro-2'-S-(4-chlorophenyl)-2'-
thiouridine (98). 89b (255 mg, 0.4 mmol) was treated with Py.9HF (920 μL, 4 mmol)
and DBH (572 mg, 2 mmol) in anhydrous CH2Cl2 (10 mL) according to general
desulfurization-difluorination procedure gave 405 mg of yellow oil after aqueous
workup. Column chromatography (10% → 70% EtOAc in hexanes) gave a major fraction
of 118 mg as a pale-yellow oil: 19F-NMR (CDCl3) δ -127.53 to -127.24 (m, 1F), -130.84
to -130.62 (m, 1F).
104
3-N-benzyl-3',5'-di-O-benzyl-5-iodo-2'-S-(4-methoxyphenyl)-2'-thiouridine (100)
and 3-N-benzyl-3',5'-di-O-benzyl-2'-fluoro-2'-S-(4-methoxyphenyl)-2'-thiouridine
(94b). NIS (95 mg, 0.42 mmol) was added to a stirred solution of 18 (128 mg, 0.2 mmol)
and Py.9HF (138 μL, 0.6 mmol) in anhydrous CH2Cl2 (3 mL) at -78 oC in a poly
propylene vessel and the resulting brown solution was brought to ~5 oC overnight (16 h).
Stirring was continued at ambient temperature for 7 h (total reaction time: 23 h). The
reaction was quenched by addition of ice-cold water (10 mL), diluted with CH2Cl2 (5
mL), neutralized with drop-wise addition of conc. NH4OH. Organic layer was separated
and aqueous layer was back extracted (2 x CH2Cl2). Combined organic layer was washed
with 1N HCl (10 mL), brine (10 mL), dried (MgSO4), and filtered. Volatiles were
evaporated in vacuo and the brown residue was column chromatographed (5% → 20%
EtOAc in hexanes) to give 100 (44 mg, 29%) as a colorless oil and 94b (16 mg, 12%) as
a pale-yellow oil (2'-R/S-S, ~ 1:1).
Analogous treatment of 18 (128 mg, 0.2 mmol) with NIS (95 mg, 0.42 mmol) and
DAST (160 μL, 1.2 mmol) followed by aqueous workup (sat. Na2S2O3, sat. NaHCO3,
brine, MgSO4) and column chromatography gave 94b (58 mg, 44%) as a pale-yellow oil
(2'-R/S-S, ~ 1:1).
100 had: 1H NMR (CDCl3) δ 7.66-7.63 (m, 2H, Ph), 7.56 (s, 1H, H6), 7.44-7.31 (m, 13H,
Ph), 7.12-7.10 (m, 2H, Ph), 6.51 (d, J = 8.9 Hz, 1H, H1'), 6.33-6.30 (m, 2H, Ph), 5.15
(“d”, J = 13.4 Hz, 1H, benzylic), 5.02 (“d”, J = 13.3 Hz, 1H, benzylic), 4.68-4.52 (m, 4H,
benzylic), 4.33-4.25 (m, 2H, H4', H3'), 3.76-3.67 (m, 2H, H2', H5'), 3.66 (s, 3H, OCH3),
3.51 (dd, J = 2.0, 10.5 Hz, 1H, H5''); 13C NMR (CDCl3) δ 159.8, 159.1, 150.8, 142.2
(C6), 137.22, 137.16, 136.2, 135.2, 130.4, 128.8, 128.6, 128.4, 128.2, 128.1, 128.06,
105
127.8, 127.7, 123.0, 114.7, 90.2 (C1'), 82.1 (C3'), 80.7 (C4'), 73.8 (CH2), 72.3 (CH2),
70.5 (C5), 68.9 (C5'), 56.4 (C2'), 55.5 (OCH3), 46.0 (CH2).
3-N-benzyl-3',5'-di-O-benzyl-5-bromo-2'-fluoro-2'-S-(4-methoxyphenyl)-2'-
thiouridine (101). Treatment of 94b (131 mg, 0.2 mmol) with DBH (172 mg, 0.6 mmol)
and Py.9HF (280 μL, 1.2 mmol) at -78 oC according to the procedure described for 89a
→ 96a/97 followed by column chromatography (15% EtOAc in hexanes) gave 101 as a
mixture of diastereomers (2'-R/S-S, ~ 65:35) (pale-yellow oil, 82 mg, 56%): 1H NMR
(CDCl3) δ 7.84 (s, 1H, H6), 7.81 (d, J = 2.1 Hz, 0.2H, Ph), 7.66 (d, J = 2.2 Hz, 0.8H, Ph),
7.49-7.23 (m, 17H, Ph), 6.78 (d, J = 8.7 Hz, 0.8H, Ph), 6.75 (d, J = 8.7 Hz, 0.2H, Ph),
6.44 (d, J = 13.9 Hz, 0.8H, H1'), 6.27 (br s, 0.2H, H1'), 5.09 (“d”, J = 13.6 Hz, 1H,
benzylic), 4.92-4.80 (m, 1H, benzylic), 4.73-4.65 (m, 1H, benzylic), 4.62-4.41 (m, 3H,
benzylic), 4.18-4.09 (m, 2H, H3', H4'), 3.92 (s, 0.6H, CH3), 3.91 (s, 2.4H, CH3), 3.82-
3.72 (m, 1H, H5'), 3.61-3.53 (m, 1H, H5''); 19F NMR (CDCl3) δ –133.52 ppm (br s, 0.8F),
–131.57 ppm (br s, 0.2F).
13C NMR (CDCl3) for major isomer (extracted data): δ 158.4, 157.2, 149.4, 138.1,
137.79, 137.76, 137.3, 136.8, 135.9, 134.1, 134.0, 129.3, 128.6, 128.5, 128.4, 128.3,
128.2, 128.1, 128.0, 127.9, 120.24, 120.23, 112.2, 112.0, 108.7 (d, 1JC2'-F = 239.1 Hz,
C2'), 96.9, 90.4 (d, 2JC1'-F = 47.3 Hz, C1'), 80.3 (C4'), 79.7 (d, 2JC3'-F = 17.7 Hz, C3'), 73.5
(CH2), 73.1 (CH2), 67.0 (C5'), 56.4 (CH3), 45.7 (CH2).
5'-O-Acetyl-2',3'-dideoxy-2',3'-didehydro-2'-[(4-chlorophenyl)sulfonyl]uridine (or)
1-[5'-O-Acetyl-2',3'-dideoxy-2'-(4-chlorophenyl)sulfonyl-β-ᴅ-glycero-pent-2'-
enofuranosyl]uracil (102). mCPBA (70% reagent, 250 mg, 1.45 mmol) was added to a
stirred solution of 89a (227.4 mg, 0.5 mmol) in CH2Cl2 (15 mL) and the resultant
106
colorless solution was stirred at ambient temperature for 12h. TLC showed complete
conversion to a more polar product. The reaction mixture was poured into NaHCO3/H2O
(10 mL). The aqueous layer was extracted with CHCl3 (2 x 5 mL) and the combined
organic phase was washed with H2O (20 mL), brine (25 mL), and dried (MgSO4).
Volatiles were evaporated in vacuo to give 91a (241 mg, 99%) as a colorless foam.
Purification of this material on silica gel chromatography (50% EtOAc in hexanes) gave
102 contaminated with 40% of 91a (184 mg, 1.5:1 ratio of 102:91a): 1H NMR (CDCl3) δ
10.04 (s, 0.6H, NH), 9.99 (s, 0.4H, NH), 7.89-7.85 (m, 0.8H, Ph), 7.84-7.81 (m, 1.2H,
Ph), 7.57-7.53 (m, 2H, Ph), 7.36 (d, J = 8.1 Hz, 0.6H, H6), 7.28-7.27 (“m”, 0.6H, H1'),
7.19 (d, J = 8.2 Hz, 0.4H, H6), 7.01 (“d”, J = 1.9 Hz, 0.6H, H3'), 6.21 (d, J = 6.4 Hz,
0.4H, H1'), 5.72-5.66 (m, 1.4H, H3', H5), 5.15-5.11 (m, 0.6H, H4'), 4.64 (t, J = 6.8 Hz,
0.4H, H2'), 4.46-4.22 (m, 2.4H, H4', H5', H5''), 2.09 (s, 1.8H, CH3), 2.08 (s, 1.2H, CH3),
2.07 (s, 1.2H, CH3).
3-N-Benzyl uracil (103) and 3,5-di-O-benzyl-1,2-dideoxy-1,2-didehydro-2-[(4-
chlorophenyl)sulfonyl]ribose (104). A stirred solution of 91b (30 mg, 0.045 mmol) and
selectfluor (24 mg, 0.067 mmol) in dry DMF (2 mL) was added to a chilled suspension of
KH (30 wt% dispersion in mineral oil; 2.5 mg, 0.0613 mmol) in dry THF (5 mL) at -78
oC. TLC after 30 minutes showed complete consumption of starting material and two new
spots. The reaction mixture was slowly warmed to 0 oC. Crude reaction mixture was
partitioned between CHCl3 (5 mL) and ice-cold NH4Cl/H2O (5 mL). The aqueous layer
was extracted with CHCl3 (2 x 2 mL) and the combined organic phase was washed with
NaHCO3/H2O (10 mL), brine (10 mL), and dried (MgSO4). Volatiles were evaporated in
107
vacuo and the residue was column chromatographed (5% → 50% EtOAc in hexanes) to
give 103 (5.7 mg, 63%) as colorless oil and 104 (14.9 mg, 71%) as a colorless foam.
Analogously, treatment of substrate (30 mg, 0.045 mmol) in dry THF (2 mL) with
a chilled suspension of KH (30 wt% dispersion in mineral oil; 2.5 mg, 0.0613 mmol) in
dry THF (5 mL) at 0 oC for 15 minutes followed by aqueous workup and column
chromatography also gave 103 and 104 in similar yields.
103 had:221 1H NMR (CDCl3) δ 9.48 (1H, br s, NH), 7.45-7.42 (m, 2H, Ph), 7.34–7.24
(m, 3H, Ph), 6.50 (dd, J = 3.7, 7.5 Hz, 1H, H6), 5.79 (d, J = 7.7 Hz, 1H, H5), 5.10 (s, 2H,
CH2).
104 had: 1H NMR (CDCl3) δ 7.71 (d, J = 8.6 Hz, 2H, Ph), 7.38 (s, 1H, H1), 7.31-7.13 (m,
10H, Ph), 6.99-6.97 (“m”, 2H, Ph), 4.81 (d, J = 2.9 Hz, 1H, H3), 4.73-4.70 (m, 1H, H4),
4.45-4.39 (m, 2H, CH2), 4.37-4.30 (m, 2H, CH2), 3.42 (dd, J = 5.4, 10.3 Hz, 1H, H5),
3.34 (dd, J = 5.7, 10.3 Hz, 1H, H5'); 13C NMR (CDCl3) δ 160.7 (C1), 140.6 (Ph), 139.4
(Ph), 137.2 (Ph), 137.0 (Ph), 129.2 (Ph), 128.9 (Ph), 128.6 (Ph), 128.4 (Ph), 128.1 (Ph),
128.0 (Ph), 127.9 (Ph), 127.8 (Ph), 118.8 (C2), 90.5 (C4), 80.6 (C3), 73.7 (CH2), 71.0
(CH2), 68.7 (C5). HRMS (ESI) m/z 493.0846 [M+Na]+, calcd for C25H23ClNaO5S+
493.0847.
[4-[(4-Methoxyphenyl)sulfonyl]furan-2-yl]methyl acetate (105), (106) and 5'-O-
Acetyl-2',3'-dideoxy-2',3'-didehydro-2'-[(4-methoxyphenyl)sulfonyl]uridine (107).
TDAE (0.24 mL, 1.04 mmol) was added to a stirred solution of 91a (100 mg, 2.07 mmol)
in DMF (2 mL) at -78 oC and the reaction mixture was brought to ambient temperature
over 1h 15 minutes and a 10% HCl solution (2 mL) was added. The crude was extracted
with EtOAc (3 x) and the combined organic phase was dried (Na2SO4). Volatiles were
108
evaporated in vacuo and the residue was column chromatographed (Petroleum ether (PE)
100% → EtOAc/PE 2/1 → 100% EtOAc) to give 105 (34.5 mg, 54%), 106 (21 mg,
24%), and 107 (35 mg, 40%) as colorless oils.
105 had: 1H NMR (CDCl3) δ 7.93 (d, J = 0.8 Hz, 1H, H1), 7.85-7.89 (m, 2H, Ph), 6.96-
7.00 (m, 2H, Ph), 6.56 (s, 1H, H3), 4.97 (s, 2H, H5, H5'), 3.85 (s, 3H, OCH3), 2.05 (s,
3H, Ac); 13C NMR (CDCl3) δ 170.3 (C=O), 163.8 (Ph), 152.4 (C4), 145.7 (C1), 132.9
(C2), 131.1 (Ph), 129.8 (Ph), 114.7 (Ph), 108.7 (C3), 57.4 (C5), 55.8 (OCH3), 20.8 (CH3);
MS (ESI) m/z 328.17 [M+NH4]+, calcd for C14H18NO6S
+ 328.08.
106 had: 1H NMR (CDCl3) δ 9.00 (br, 1H, NH), 7.76 (d, J = 8.8 Hz, 2H, Ph), 7.58 (s, 1H,
H1'), 6.97-6.99 (m, 3H, Ph, H6), 5.69 (br s, 1H, OH), 5.62 (dd, J = 1.2, 8 Hz, 1H, H5),
4.93-4.79 (m, 1H, H3'), 4.27 (dd, J = 4.4, 12.4 Hz, 1H, H5'), 4.21 (dd, J = 4.8, 12.4 Hz,
1H, H5''), 3.86 (s, 3H, OCH3), 1.95 (s, 3H, Ac); 13C NMR (CDCl3) δ 170.4 (C=O), 164.2
(Ph), 162.6 (C4), 160.3 (C6, C2), 150.3 (C4’), 140.2 (C2'), 131.5 (Ph), 129.9 (Ph), 116.9
(C1'), 114.9 (Ph), 103.7 (C5), 88.7 (C3'), 63.4 (C5'), 55.6 (OCH3), 20.5 (CH3). MS (ESI)
m/z 440.04 [M+NH4]+, calcd for C18H22N3O8S
+ 440.11.
107 had: 1H NMR (CDCl3) δ 9.23 (br, 1H, NH), 7.79-7.75 (m, 2H, Ph), 7.24 (d, J = 8.4
Hz, 1H, H6), 7.18 (t, J = 1.6 Hz, 1H, H3'), 7.03-6.99 (m, 3H, Ph, H1'), 5.55 (dd, J = 2.0,
8.4 Hz, 1H, H5), 5.12-5.09 (m, 1H, H4'), 4.41 (dd, J = 4.0, 12.4 Hz, 1H, H5'), 4.29 (dd, J
= 3.6, 12.4 Hz, 1H, H5''), 3.87 (s, 3H, OCH3), 2.09 (s, 3H, Ac); 13C NMR (CDCl3) δ
170.1 (C=O), 164.9 (Ph), 162.9 (C4), 150.3 (C2), 142.1 (C6), 141.9 (C2'), 139.5 (C3'),
130.8 (Ph), 129.1 (Ph), 115.2 (Ph), 103.2 (C5), 87.5 (C1'), 82.8 (C4'), 63.9 (C5'), 56.1
(OCH3), 20.8 (Ac). HRMS (ESI) m/z 423.0856 [M+H]+, calcd for C18H19N2O8S+
423.0857.
109
[4-[(4-Methoxyphenyl)sulfonyl]furan-2-yl]methyl acetate (105) and uracil (105a). In
a glove box, SED (31 mg, 0.1 mmol) was added to a solution of 91a (50 mg, 0.1 mmol)
in anhydrous DMF (4 mL) and stirred at ambient temperature for 15 minutes. Reaction
flask was brought out of the glove box and water was added to the crude reaction
mixture. The aqueous phase was extracted with CH2Cl2 (3 x), the combined organic
phase was dried with Na2SO4 and volatiles were evaporated in vacuo to give 105 (29.4
mg, 95%) as a colorless oil.
Acidification of the aqueous phase (pH ~ 5-6) followed by a second extraction
with CH2Cl2 (2 x) gave a white solid. 1H NMR (MeOD) of the white solid revealed the
presence of uracil 105a along with other impurities.
3,5-di-O-Benzyl-1,2-dideoxy-1,2-didehydro-2-[(4-methoxyphenyl)sulfonyl]ribose
(108), 2-[(benzyloxy)methyl]-4-[(4-methoxyphenyl)sulfonyl]furan (109) and 3-N-
benzyluracil (103). In a glove box, SED (22 mg, 0.067 mmol) was added to a stirred
solution of 91b (45 mg, 0.07 mmol) in DMF (4 mL) and the reaction mixture was stirred
at ambient temperature for 15 minutes. Reaction flask was brought out of the glove box
and water was added to the crude reaction mixture. The aqueous phase was extracted
with CH2Cl2 (3 x) and the combined organic phase was dried with Na2SO4. Volatiles
were evaporated in vacuo and the residue was column chromatographed (PE 100% →
PE/EtOAc 9/1 → EtOAC 100%) to give 108 (22 mg, 70%) as a white gum and 103 (9.4
mg, 69%) as a white solid.
108 had: 1H NMR (CDCl3) δ 7.83-7.79 (m, 2H, Ph), 7.42 (s, 1H, H1), 7.37-7.31 (m, 3H,
Ph), 7.26-7.24 (m, 5H, Ph), 7.09-7.07 (m, 2H, Ph), 6.86-6.84 (m, 2H, Ph), 4.86 (d, J = 3.2
Hz, 1H, H3), 4.77 (td, J = 3.2, 5.6 Hz, 1H, H4), 4.49 (d, J = 2.4 Hz, 2H, CH2), 4.40 (s,
110
2H, CH2), 3.81 (s, 3H, OCH3), 3.48 (dd, J = 5.6, 10.4 Hz, 1H, H5), 3.40 (dd, J = 5.6, 10.4
Hz, 1H, H5); 13C NMR (CDCl3) δ 163.3 (CPh), 159.7 (C1), 137.4 (CPh), 137.3 (CPh),
133.6 (CPh), 129.8 (2 CHPh), 128.7 (2 CHPh), 128.4 (2 CHPh), 128.1 (3 CHPh), 128.0
(CHPh), 127.9 (2 CHPh), 119.7 (C2), 114.2 (2 CHPh), 90.3 (C3), 80.7 (C4), 73.7 (CH2),
70.8 (CH2), 68.9 (C5), 55.7 (OCH3); HRMS (ESI) m/z 484.1789 [M+NH4]+, calcd for
C26H30NO6S+ 484.1788.
Analogous treatment of and 91b (50 mg, 0.075 mmol) with SED (64 mg, 0.22
mmol) in DMF (4 mL) for 30 minutes at room temperature gave 109 as a mixture with
108 (7.4 mg, 2:1 ratio of 109:108) as a colorless oil and 103 (13 mg, 86%) as a white
solid.
109 had: 1H NMR (200MHz, CDCl3) δ 7.93 (s, 1H, H1), 7.91-7.84 (m, 2H, Ph), 7.36-
7.30 (m, 5H, Ph), 7.01-6.96 (m, 2H, Ph), 6.49 (s, 1H, H3), 4.53 (s, 2H, CH2), 4.42 (s, 2H,
CH2), 4.14-4.03 (m, 2H, H5), 3.86 (s, 3H, OCH3); MS (ESI) m/z 376.17 [M+NH4]+, calcd
for C19H22NO5S+ 376.12.
5'-O-Acetyl -2',3'-didehydro-2',3'-dideoxy-2'-fluorouridine (111) and 1-(5-O-Acetyl-
2-deoxy-2-fluoro-β,D-arabinofuranosyl)uracil (114). TDAE (0.06 mL, 50.1 mg, 0.25
mmol) was added drop-wise via syringe to a stirred solution of 95a (95 mg, 0.19 mmol)
in dry THF (5 ml) at -70°C under sunlamp irradiation. After 1 h, Selectfluor (131 mg,
0.37 mmol) was added and the sunlamp was turned off. The resulting mixture was stirred
for 1 h at -70°C and then overnight at ambient temperature. The reaction mixture was
quenched with 3 ml of aqueous NH4Cl and stirred for 30 min and then extracted with
EtOAc. The combined organic layer was washed with Na2CO3/H2O, brine and dried over
111
MgSO4. Evaporation and column chromatography (5% MeOH in CHCl3) gave 111 (10
mg: 18%) and 114 (5 mg: 10%).
111 was also obtained by another procedure (see Scheme 22). In a glove box,
SED (68 mg, 0.24 mmol, 3 equiv.) was added to a stirred solution of 95a (40 mg, 0.08
mmol) in anhydrous DMF (4 mL) and the resulting dark brown solution was stirred at
120 oC for 18 h. Reaction flask was brought out of the glove box, and 10% HCl solution
was added to the crude reaction mixture. Aqueous layer was extracted with CH2Cl2 (2 x).
Combined organic layer was dried (Na2SO4), filtered, and concentrated in vacuo to give a
brown solid. Et2O was added to the solid and was filtered. Filtrate was concentrated in
vacuo to give 111 (<10 mg, ~ 46%) along with other impurities. 1H NMR of the black
solid (DMSO-d6) showed similar peaks to the ones obtained for the oxidized form of the
SED (SED2+).
111173 had: 1H NMR (CDCl3) δ 8.47 (br s, 1H, NH), 7.52 (dd, J = 1.0, 8.1 Hz, 1H, H6),
6.90-6.88 (m, 1H, H1'), 5.78 (d, J = 8.1 Hz, 1H, H5), 5.69 (m, 1H, H3'), 5.06-5.02 (m,
1H, H4'), 4.35-4.21 (m, 2H, H5',5"), 2.11 (s, 3H, Ac); 19F NMR (CDCl3) δ -133.76 (t, J =
4.6 Hz, F2'); MS (EI) m/z 271.09 [M+H]+. calcd for C11H12FN2O5+ 271.07.
114 had: 1H NMR (CDCl3) δ 8.32 (br s, 1H, NH), 7.51-7.53 (m, 1H, H6), 6.22-6.28 (m,
1H, H1'), 5.76 (d, J = 8.1 Hz, 1H, H5), 5.06 (d, J = 51.3 Hz, 1H, H2'), 4.43-4.29 (m, 3H,
H3', H5', H5''), 4.21-4.19 (m, 1H, H4'), 2.12 (s, 3H, Ac); 13C NMR (150 MHz, CDCl3) δ
170.9 (C=O), 162.7 (C4), 150.1 (C2), 140.9 (C6), 102.2 (C5), 94.4 (d, J = 191.8 Hz,
C2’), 84.7 (d, J = 16.5 Hz, C1’), 82.9 (C4’), 75.4 (d, J = 26.5 Hz, C3’), 63.1 (C5’), 20.9
(CH3); 19F NMR (CDCl3) δ -199.91 (s, F2'); MS (EI) m/z 288.9 [M+H]+, calcd for
C11H14FN2O6+ 289.08.
112
3-(benzyloxy)-2-[(benzyloxy)methyl]-4-[(4-methoxyphenyl)sulfonyl]furan (112). In a
glove box, SED (62 mg, 0.22 mmol) was added to a solution of 95b (50 mg, 0.073 mmol)
in anhydrous DMF (4 mL) and the resulting solution was stirred at 120 oC for 3 h.
Reaction flask was brought out of the glove box, and water was added to the crude
reaction mixture. The aqueous phase was extracted with CH2Cl2 (3 x) and the combined
organic phase was dried with Na2SO4. Volatiles were evaporated in vacuo and the residue
was column chromatographed (PE/EtOAc 9/1 → EtOAC 100%) to give 112 (19.6 mg,
58%) as a white gum and 103 (12.4 mg, 84%) as a white solid.
Analogous treatment of 95b (58.3 mg, 0.085 mmol) with SED (72 mg, 0.25
mmol) at ambient temperature for 4 h showed only trace amounts of 112 and 103 on
TLC. Addition of degazed water (0.5 mL, 500 mg, 27.75 mmol) followed by heating at
80 oC for 30 minutes gave, after workup and silica gel chromatography, 112 (31.7 mg,
80%) and 103 (10.1 mg, 60%).
112 had: 1H NMR (200MHz, CDCl3) δ 7.94-7.87 (m, 2H, Ph), 7.83 (s, 1H, H1), 7.37-
7.24 (m, 10H, Ph), 6.95-6.88 (m, 2H, Ph), 5.07 (s, 2H, CH2), 4.41 (s, 2H, CH2), 4.17 (s,
2H, CH2), 3.84 (s, 3H, OCH3); 13C NMR (50 MHz, CDCl3) δ 163.6 (C3), 144.8 (C1),
142.4 (Ph), 140.7 (Ph), 137.4 (Ph), 136.2 (Ph), 132.8 (C4), 130.0 (Ph), 128.55(Ph),
128.53 (Ph), 128.48 (Ph), 128.0 (Ph), 127.9 (Ph), 125.6 (C2), 114.3 (Ph), 77.3 (CH2),
72.4 (CH2), 61.1 (CH2), 55.7 (OCH3). HRMS (ESI) m/z 482.1632 [M+NH4]+ calcd for
C26H28NO6S+ 482.1632.
3-N-Methyl-2'-deoxy-2'-fluoro-2'-[(4-methoxyphenyl)sulfonyl]uridine (115). TDAE
(0.036 mL, 0.155 mmol) was added drop-wise via syringe to a stirred solution of 95a
(15.4 mg, 0.031 mmol) in dry THF or DMF (0.5 ml) at -70°C under N2. UV sunlamp
113
was switched on and after 1 h, CH3I (44.4 mg, 0.31 mmol) was added. The resulting
mixture (yellowish with white fine solid) was stirred for 1 h at -70°C and then overnight
at ambient temperature. The reaction mixture was quenched with 3 ml of aqueous MeOH,
stirred for 30 min and concentrated in vacuo. The crude product (100.8 mg), a yellowish
solid, was washed with dichloromethane (2 x 5 mL), filtered and the filtrate concentrated
in vacuo to recover 42.9 mg of a viscous orange. 19F-NMR of this crude product
indicated no starting material (a triplet centred at - 157 ppm) and a triplet centred at -
161.42 ppm with additional resonances at in the region -122 to -126 ppm. Column
chromatography (5% MeOH in CHCl3) gave 115 (10.2 mg, ~ 79%) that was
contaminated with an impurity, as the fastest moving fraction.
1H NMR (300 MHz, CDCl3) δ 7.75 (d, 2H, J = 6.9 Hz, Ph), 7.68 (d, 1H, J = 8.7 Hz, H6),
6.98 (d, 2H, J = 6.9 Hz, Ph), 6.40 (d, 1H, J = 20.4 Hz, H1'), 5.85 (d, 1H, J = 8.7 Hz, H5),
5.04 (m, 1H, H3'), 3.98-4.11 (m, 3H, H4' and H5',5''), 3.89 (s, 3H, OCH3); 3.15 (s, 3H,
NCH3); 19F NMR (282 MHz, CDCl3) δ -160.41 (t, J = 19.5 Hz, F2') and -160.75 (t, J =
19.8 Hz, F2') (ratio 1:0.16); MS (ESI) m/z 431.1 [M+ H]+, 453.1 [M+Na]+.
General Procedure for cyclic voltammetry. Electrochemical measurements were
performed using an EG & G-Princeton Applied Research 263A all-in-one potentiostat,
using a standard three-electrode setup with a glassy carbon electrode (working electrode,
diameter = 3 mm), platinum wire auxiliary electrode and a non-aqueous Ag/Ag+ (0.01 M
AgNO3 + 0.1 M n-Bu4NClO4) system in acetonitrile as the reference electrode. All
solutions under the study were 0.1 M in the supporting electrolyte n-Bu4NPF6 (Fluka
puriss electrochemical grade) with the voltage scan rate of 0.2 V s-1. Anhydrous DMF
was obtained from Fisher Scientific. Solutions (2.5 mL) were thoroughly bubbled with
114
dry argon for 15 minutes to remove oxygen before any experiment and kept under
positive pressure of argon. Under these experimental conditions the ferrocene/ferricinium
couple, used as internal reference for potential measurements, was located at E1/2 = + 0.05
V in DMF.
4-Benzylamino-7-(β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (122a). BnBr (345 μL,
496 mg, 2.9 mmol) was added to a stirred solution of tubercidin (19a; 266 mg, 1 mmol)
in dried DMF (5 mL). After stirring for 48 hours at 40 oC, TLC showed almost complete
conversion to a more polar product. Volatiles were evaporated to ~1 mL (< 40 oC,
vacuum pump) and the resulting syrup was added dropwise to dried acetone (30 mL) with
vigorous stirring. Et2O (60 mL) was added to the suspension, which was chilled for 20
min at 0 oC and vacuum filtered. The hygroscopic precipitate was quickly dissolved in
MeOH (10 mL) and Me2NH/THF (2 M, 8 mL) was added. The resulting solution was
stirred at reflux (65 ˚C, oil bath) for 20 h. TLC showed ~80% conversion to less polar
spot, and volatiles were evaporated and coevaporated with MeOH (2 ×). The residue was
dissolved in warm MeOH (10 mL), H2O (60 mL) was added, and the solution was
extracted with EtOAc (5 × 20 mL). The combined organic phase was dried (Na2SO4),
concentrated in vacuo, coevaporated (2 × EtOH) and flash chromatographed (EtOAc) to
give 122a (238 mg, 67%) as a colorless oil: UV (MeOH) λmax 276 nm , λmin 244 nm; 1H
NMR (DMSO-d6) δ 8.09-8.13 (m, 2H, NH, H2), 7.38 (d, J = 3.7 Hz, 1H, H6), 7.29-7.35
(m, 4H, Ph), 7.22-7.25 (m, 1H, Ph), 6.67 (d, J = 3.4 Hz, 1H, H5), 6.01 (d, J = 5.9 Hz, 1H,
H1'), 5.27-5.32 (m, 2H, 2'-OH, 5'-OH), 5.12 (d, J = 4.3 Hz, 1H, 3'-OH), 4.70-4.78 (m,
2H, CH2), 4.44 (q, J = 5.7 Hz, 1H, H2'), 4.09 (“q”, J = 4.1 Hz, 1H, H3'), 3.90 (q, J = 3.4
Hz, 1H, H4'), 3.60-3.65 (m, 1H, H5'), 3.50-3.56 (m, 1H, H5''); 13C NMR (DMSO-d6) δ
115
156.1, 151.4, 149.5, 140.1, 128.2 (Ph), 127.1 (Ph), 126.6, 122.3 (C6), 103.4, 99.2 (C5),
87.6 (C1'), 85.1 (C4'), 73.7 (C2'), 70.7 (C3'), 61.8 (C5'), 43.1 (CH2); MS (ESI) m/z 357
(100%, MH+), HRMS (ESI) m/z 357.1581 (MH+), calcd for C18H29N4O4 357.1563.
4-(4-Nitrobenzylamino)-7-(β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (122b).
Method A. Tubercidin (19a; 266 mg, 1.0 mmol) was treated with 4-nitrobenzyl bromide
(648 mg, 3.0 mmol) at 80 ˚C for 24 h as described above for 122a followed by addition
of MeOH (15 mL) and Me2NH/THF (2 M, 8 mL). The reaction mixture was stirred at
reflux for 20 h; TLC showed ~85% conversion to a less polar product 122b (244 mg,
56%, yellow oil): UV (MeOH) λmax 278 nm, λmin 240 nm; 1H NMR (DMSO-d6) δ 8.30 (t,
J = 6.1 Hz, 1H, NH), 8.19 (“d”, J = 8.8 Hz, 2H, Ph), 8.12 (s, 1H, H2), 7.58 (d, J = 8.8 Hz,
2H, Ph), 7.43 (d, J = 3.7 Hz, 1H, H6), 6.69 (d, J = 3.5 Hz, 1H, H5), 6.04 (d, J = 6.3 Hz,
1H, H1'), 5.28-5.30 (m, 2H, 2 × OH), 5.14 (s, 1H, OH), 4.80-4.91 (m, 2H, CH2), 4.45
(“d”, J = 4.4 Hz, 1H, H2'), 4.11- 4.13 (m, 1H, H3'), 3.92 (q, J = 3.5 Hz, 1H, H4'), 3.64
(dt, J = 3.9, 11.8 Hz, 1H, H5'), 3.52-3.57 (m, 1H, H5''); 13C NMR (DMSO-d6-D2O) δ
155.8, 151.0, 148.8, 147.9, 146.3, 127.9 (Ph), 123.4 (Ph), 122.8 (C6), 103.6, 99.3 (C5),
87.5 (C1'), 84.9 (C4'), 73.5 (C2'), 70.4 (C3'), 61.6 (C5'), 42.7 (CH2); MS (ESI): m/z 402
(100%, MH+). Anal. Calcd for C18H19N5O6•1.25 H2O (423.89): C, 51.00; H, 5.11; N,
16.52. Found: C, 51.05; H, 5.06; N, 16.03.
Method B. Step a. Ac2O (377 μL, 408 mg, 4 mmol) was added to a stirred suspension of
19a (266 mg, 1 mmol) in dried pyridine (5 mL) at 0 oC (ice bath) and stirring was
continued at 0 oC for 12 h and then at ambient temperature for 9 h (total reaction time: 21
h). MeOH was added, the reaction mixture was stirred at ambient temperature for 30 min,
and volatiles were evaporated (vacuum pump, <25 oC). MeOH was added and
116
evaporated, and the resulting gum was partitioned between CHCl3 (50 mL) and 2%
AcOH/H2O (50 mL). The aqueous layer was extracted with CHCl3, and the combined
organic phase was washed with NaHCO3/H2O, brine, and dried (MgSO4). Volatiles were
evaporated in vacuo and the residue was column chromatographed (EtOAc) to give
2',3',5'-tri-O-acetyltubercidin (123; 337 mg, 86%) as a colorless foam with data as
reported.222
Step b. NaNO2 (66 mg, 0.95 mmol) was added to a solution of 123 (150 mg, 0.38 mmol)
in freshly prepared ~55% HF-pyridine192 (1.9 mL) at –10 oC in a sealed polypropylene
vessel. The mixture was stirred at –10 oC for 15 min, and TLC showed almost complete
conversion to a less polar spot. Ice/H2O was added and the mixture was extracted with
CH2Cl2. The combined organic phase was washed with NaHCO3/H2O, brine, and dried
(Na2SO4). Volatiles were removed in vacuo, and the resulting brown oil was column
chromatographed (30% EtOAc in hexanes) to give 7-(2,3,5-tri-O-acetyl-β-D-
ribofuranosyl)-4-fluoropyrrolo[2,3-d]pyrimidine (124; 124 mg, 82%) as a colorless oil:
UV (MeOH) λmax 258 nm, λmin 233 nm; 1H NMR (CDCl3) δ 8.53 (d, 4JH-F = 0.5 Hz, 1H,
H2), 7.37 (d, J = 3.8 Hz, 1H, H8), 6.66 (d, J = 3.8 Hz, 1H, H7), 6.45 (d, J = 6.0 Hz, 1H,
H1'), 5.74 (t, J = 5.8 Hz, 1H, H2'), 5.55 (dd or m, J = 4.1, 5.6 Hz, 1H, H3'), 4.32-4.42 (m,
3H, H4', H5', H5''), 2.12 (s, 3H, CH3), 2.13 (s, 3H, CH3), 2.02 (s, 3H, CH3); 19F NMR
(CDCl3) δ –64.81 ppm (s); 13C NMR (CDCl3) δ 170.2, 169.6, 169.4 (3 x C=O), 162.3 (d,
1JC-F = 253.8 Hz, C4), 155.1 (d, 3JC-F = 11.9 Hz, C7a), 151.1 (d, 3JC-F = 14.5 Hz, C2),
125.7 (d, 4JC-F = 2.6 Hz, C6), 105.4 (d, 2JC-F = 33.4 Hz, C4a), 99.6 (d, 3JC-F = 4.9 Hz, C5),
86.1 (C1'), 79.9 (C4'), 73.1 (C2'), 70.1 (C3'), 63.3 (C5'), 20.7, 20.5, 20.3 (3 x s, 3 x CH3);
MS (ESI): m/z 396 (100%, MH+).
117
Step c. Freshly distilled Et3N (113 μL, 82 mg, 0.81 mmol) was added to a stirred
suspension of 124 (93 mg, 0.23 mmol) and 4-nitrobenzylamine hydrochloride (67 mg,
0.35 mmol) in MeOH (3 mL) and stirring was continued for 5 h at ambient temperature.
Volatiles were evaporated in vacuo and the residue was column chromatographed (50%
EtOAc in hexanes) to give 2',3',5'-tri-O-acetyl-4-N-(4-nitrobenzyl)tubercidin (125; 58
mg, 47%) as a colorless oil: UV (MeOH) λmax 277 nm, λmin 238 nm. 1H NMR (CDCl3) δ
8.36 (s, 1H, H2), 8.17 (d, J = 8.7 Hz, 2H, Ph), 7.51 (d, J = 8.7 Hz, 2H, Ph), 7.12 (d, J =
3.768 Hz, 1H, H6), 6.42-6.46 (dd, 3.8, 6.0 Hz, 2H, H1', H5), 5.73 (t, J = 5.8 Hz, 1H, H2'),
5.54-5.59 (m, 2H, H3', NH), 4.95 (d, J = 6.1 Hz, 2H, CH2), 4.31-4.40 (m, 3H, H4', H5',
H5''), 2.14 (s, 6H, 2 × CH3), 2.04 (s, 3H, CH3); 13C NMR (CDCl3) δ 170.4, 169.7, 169.5
(3 x C=O), 156.0 (C4), 152.3 (C2), 150.8 (C7a), 147.3 (Ph), 146.7 (Ph), 128.0 (Ph), 123.9
(Ph), 121.5 (C6), 103.8 (C4a), 99.4 (C5), 85.4 (C1'), 79.5 (C4'), 73.1 (C2'), 70.8 (C3'),
63.5 (C5'), 44.2 (CH2), 20.8, 20.6, 20.4 (3 x s, 3 x CH3). MS (ESI): m/z 528 (100%,
MH+).
Step d. NH3/MeOH (5 mL) was added to a stirred solution of 125 (54 mg, 0.1 mmol) in
MeOH (1 mL) and stirring was continued at ambient temperature for 20 h. Volatiles were
evaporated and the residue was column chromatographed with a 2 → 4% gradient of the
upper phase of EtOAc/i-PrOH/H2O (4:1:2) in EtOAc to give 122b (35 mg, 85%) as a
yellow oil.
5-Carboxamido-4-(4-nitrobenzylamino)-7-(β-D-ribofuranosyl)pyrrolo[2,3-
d]pyrimidine (122c). Sangivamycin (19b; 155 mg, 0.5 mmol) was treated with 4-
nitrobenzyl bromide (162 mg, 1.5 mmol) for 26 h at 40 oC as described for 19a → 122a
and the alkylated intermediate was then stirred with Me2NH/THF (2 M, 6 mL) and
118
MeOH (12 mL) for 45 h at ambient temperature. Me2NH/THF (2 M, 2 mL) was added
and the mixture was stirred at reflux for 32 h (total reaction time: 77 h). TLC showed
~80% conversion to the less polar 122c, which was obtained as off-white crystals (101
mg, 45%) after recrystallization from EtOH: mp 154-158 oC (dec.); UV (MeOH) 286 nm
(ε 23 100), λmin 229 nm (ε 8700); 1H NMR (DMSO-d6) δ 10.29 (t, J = 6.0 Hz, 1H, NH),
8.17-8.21 (m, 4H, Ph, H2, H6), 8.11 and 7.47 (2 × s, 2 x 1H, CONH2), 7.59 (brd, J = 8.8
Hz, 2H, Ph), 6.06 (d, J = 6.0 Hz, 1H, H1'), 5.43 (d, J = 6.3 Hz, 1H, 2'-OH), 5.20 (d, J =
5.0 Hz, 1H, 3'-OH), 5.09 (t, J = 5.8 Hz, 1H, 5'-OH), 4.90 (“d”, J = 6.2 Hz, 2H, CH2), 4.37
(q, J = 5.8 Hz, 1H, H2'), 4.10 (“q”, J = 4.5 Hz, 1H, H3'), 3.93 (q, J = 4.0 Hz, 1H, H4'),
3.61-3.67 (m, 1H, H5'), 3.53-3.58 (m, 1H, H5''); 13C NMR (DMSO-d6) δ 166.5, 156.5,
152.6, 150.4, 148.0, 146.4, 128.0 (Ph), 125.7, 123.6, 110.9, 101.7, 87.2 (C1'), 85.3 (C4'),
73.9 (C2'), 70.5 (C3'), 61.9 (C5'), 42.9 (CH2); MS (ESI) m/z 445 (100%, MH+). Anal.
Calcd for C19H20N6O7•1.5 H2O (471.42): C, 48.41; H, 4.92; N, 17.83. Found: C, 48.59;
H, 4.66; N, 17.65.
5-Cyano-4-(4-nitrobenzylamino)-7-(β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine
(122d). Toyocamycin (19c; 146 mg, 0.5 mmol) was heated with 4-nitrobenzyl bromide
(364 mg, 1.5 mmol) in dried DMF (3 mL) at 40 oC for 63 h (TLC showed ~85%
conversion to a less polar product). Volatiles were evaporated to ~1 mL (< 40 oC,
vacuum pump) and this material was added dropwise to 20 mL of vigorously stirred dried
acetone. Et2O (40 mL) was added to the stirred suspension, which was chilled at 0 oC for
20 minutes and the precipitate was collected by vacuum filtration. Recrystallization from
MeOH gave 122d (99 mg, 46%) as colorless crystals: mp 214-216 oC; UV (MeOH) λmax
274, 236 nm (ε 22 100, 18 900), λmin 250, 228 nm (ε 12 800, 18 000); 1H NMR (DMSO-
119
d6) δ 8.35 (s, 1H, H2), 8.19-8.23 (m, 3H, H6, Ph), 7.59 (d, J = 8.6 Hz, 2H, Ph), 6.87 (s,
1H, NH), 5.93 (d, J = 5.6 Hz, 1H, H1'), 5.47 (d, J = 6.0 Hz, 1H, 2'-OH), 5.35 (“s”, 2H,
CH2), 5.21 (d, J = 5.0 Hz, 1H, 3'-OH), 5.09 (t, J = 5.4 Hz, 1H, 5'-OH), 4.31 (q, J = 5.5
Hz, 1H, H2'), 4.08 (q, J = 4.6 Hz, 1H, H3'), 3.92 (q, J = 3.7 Hz, 1H, H4'), 3.62-3.68 (m,
1H, H5'), 3.53-3.58 (m, 1H, H5''); 13C NMR (DMSO-d6-D2O) δ 152.6, 149.4, 146.7,
144.9, 142.7, 129.4, 128.5 (Ph), 123.5 (Ph), 115.2, 105.0, 87.7 (C1'), 85.8 (C4'), 85.5,
74.6 (C2'), 70.1 (C3'), 61.1 (C5'), 48.8 (CH2); MS (ESI): m/z 427 (100%, MH+). Anal.
Calcd for C19H18N6O6•0.5 H2O (435.39): C, 52.41; H, 4.40; N, 19.30. Found: C, 52.17;
H, 4.29; N, 19.30.
8-azidotoyocamycin (129). NaN3 (158.1 mg, 2.43 mmol) was added to a stirred solution
of 55 (300 mg, 0.810 mmol) in anhydrous DMF (10 mL) in a flame dried flask and the
resulting colorless solution was stirred at room temperature in the dark for 16 h, by which
time the solution turned a brown-red color and UV showed 6 nm bathochromic shift in
the λmax, indicating the conversion to product. Volatiles were removed from the clear,
brown-red solution using high vacuum rotary evaporator on a hot water bath (<80 oC)
while in a dark room. The resulting brown gummy oil was purified using column
chromatography in the dark (5%, MeOH/EtOAc) to give 129 as an off white solid (123.2
mg, 46%): HRMS (ESI): m/z [M+Na]+ calcd for C12H12N8NaO4
+: 355.0874; found
355.0952.
Click product (132). Cyclooctyne 130 (9.04 mg, 0.060 mmol) was added to a stirred
solution of 129 (20 mg, 0.060 mmol) in ACN and H2O (3:1) in a flask and the resulting
clear, colorless solution was stirred at room temperature for 4 h, by which time TLC
showed >80% conversion to product to a more polar product. Volatiles were removed
120
using high vacuum rotary evaporator and a hot water bath (<80 oC). The resulting yellow
oil was purified using column chromatography (10%, EtOAc/MeOH) giving 132 as an
off white solid (17 mg, 59%). This solid was then re-purified using RP-HPLC (20% CAN
in H2O) to give 132 as an off white gummy solid (9.6 mg, 33%). Rf (EtOAc:iPrOH:H2O,
4:1:2) 0.49; HRMS (ESI): m/z [M+H]+ calcd for C22H27N8O5
+: 483.2099; found
483.2087.
Click product (133). Cyclooctyne 131 (16.6 mg, 0.060 mmol) was added to a stirred
solution of 129 (20 mg, 0.060 mmol) in ACN and H2O (3:1) in a flask and the resulting
clear, pale red solution was stirred at room temperature for 4 h, by which time TLC
showed >80% conversion to product to a more polar product almost on the baseline.
Volatiles were removed using high vacuum rotary evaporator and a hot water bath (<80
oC). The resulting pale red oil was purified using column chromatography (17.5%,
EtOAc/MeOH) giving 133 as an off white gummy solid (17.1 mg, 47%). Rf
(EtOAc:iPrOH:H2O, 4:1:2) 0.13; HRMS (ESI): m/z [M+H]+ calcd for C30H29N10O5
+:
609.2317; found 609.2352.
2',3',5'-tri-O-acetyltoyocamycin. (138). Ac2O (380μL, 4 mmol) was added to a stirred
solution of 19c (292 mg, 1 mmol) in anhydrous pyridine (5 mL) at 0 oC (ice bath) and
stirring was continued at 0 oC for 1 h and then at ambient temperature for 3 h (total
reaction time: 4 h). MeOH was added, the reaction mixture was stirred at ambient
temperature for 30 min, and volatiles were evaporated (vacuum pump, <25 oC). MeOH
was added and evaporated, and the resulting gum was partitioned between CHCl3 (25
mL) and 2% AcOH/H2O (25 mL). The aqueous layer was extracted with CHCl3, and the
combined organic phase was washed with NaHCO3/H2O, brine, and dried (MgSO4).
121
Volatiles were evaporated in vacuo and the residue was column chromatographed (70%
EtOAc in hexanes) to give 2',3',5'-tri-O-acetyltoyocamycin (138; 405 mg, 97%) as a
colorless foam: 1H NMR (CDCl3) δ 8.24 (s,1H, H2), 7.72 (s, 1H, H8), 6.37 (s, 2H, NH2),
6.30 (d, J = 5.4 Hz, 1H, H1'), 5.66 (d, J = 5.5 Hz, H2'), 5.48 (d, J = 5.1 Hz, H3'), 4.38
(“q”, J = 3.9 Hz, H4'), 4.33-4.32 (“m”, 2H, H5', H5''), 2.08 (s, 3H, CH3), 2.06 (s, 3H,
CH3), 2.00 (s, 3H, CH3).
8-Bromo-2',3',5'-tri-O-acetyltoyocamycin (139). DBH (157 mg, 0.55 mmol) was added
to a stirred solution of 138 (209 mg, 0.5 mmol) in CH2Cl2 (4 mL) and the resulting brown
solution was stirred at ambient temperature for 18 hours. The reaction mixture was
partitioned between CH2Cl2 (16 mL) and NaHCO3/H2O (25 mL). The aqueous layer was
extracted with CH2Cl2, and the combined organic phase was washed with brine and dried
(MgSO4). Volatiles were evaporated in vacuo and the residue was column
chromatographed (50% EtOAc in hexanes) to give 139 (195 mg, 79%) as a brown oil
with data as reported.223
2',3',5'-tri-O-(tert-butyldimethylsilyl)toyocamycin (141). Imidazole (1.02 g, 15 mmol)
and TBDMSCl (1.09 g, 7.2 mmol) was added to a stirred solution of 19c (583 mg, 2
mmol) in anhydrous DMF (6 ml) and the resulting suspension was stirred at ambient
temperature for 24 h and then at 35 oC for 12 h, by which time TLC showed ~40%
unchanged substrate and two less polar spots. A second portion of imidazole (1.02 g, 15
mmol) and TBDMSCl (1.09 g, 7.2 mmol) were added and the resulting suspension was
stirred at 35 oC for 24 h (total reaction time: 60 h), by which time TLC showed 80%
conversion to a major less polar spot. Volatiles were evaporated and co-evaporated with
toluene (high vacuum rotavap, <40 oC). Crude pale gum was partitioned between CHCl3
122
(50 mL) and brine (50 mL). The aqueous layer was extracted with CHCl3, and the
combined organic layer was dried over anhydrous MgSO4. Volatiles were evaporated in
vacuo and the residue was column chromatographed (25% EtOAc in hexanes) to give
141 (736 mg, 58%) as a colorless oil: 1H NMR (CDCl3) δ 8.30 (s,1H, H2), 8.13 (s, 1H,
H8), 6.27 (s, 2H, NH2), 6.18 (d, J = 4.1 Hz, 1H, H1'), 4.32 (d, J = 4.2 Hz, H2'), 4.21 (d, J
= 4.4 Hz, H3'), 4.12-4.10 (“m”, 1H, H4'), 4.02 (dd, J = 2.8, 11.6 Hz, 1H, H5'), 3.78 (dd, J
= 1.8, 11.6 Hz, 1H, H5''), 0.97 (s, 9H, tBut), 0.89 (s, 9H, tBut), 0.80 (s, 9H, tBut), 0.16 (s,
3H, CH3), 0.15 (s, 3H, CH3), 0.06 (s, 3H, CH3), 0.05 (s, 3H, CH3), -0.04 (s, 3H, CH3), -
0.14 (s, 3H, CH3).
8-Iodo-2',3',5',-tri-O-(tert-butyldimethylsilyl)toyocamycin (142). LDA (2 M solution
in THF, 560 μL, 1.1 mmol) was added to a stirred solution of 141 (139.5 mg, 0.22 mmol)
in dry THF (2 mL) at -78 oC. After ~1 h at -78 oC, iodine (84 mg, 0.33 mmol). The
resultant brown solution was stirred at -78 oC for 2 h and at ambient temperature for 48
hours (total reaction time: 51h). Reaction mixture was portioned between EtOAc (10 mL)
and 2% AcOH/H2O (10 mL). Aqueous layer was extracted with EtOAc. Combined
organic layer was washed with brine (20 mL), dried (MgSO4), filtered and concentrated
in vacuo, and column chromatographed (10% EtOAc in hexanes) to give 142 (27.3 mg,
16%) and 141 (82.3 mg, 58%) as brown oils.
142 had: 1H NMR (CDCl3) δ 8.40 (s,1H, H2), 8.07 (s, 1H, NH2), 6.15 (d, J = 4.5 Hz, 1H,
H1'), 4.78 (s, 1H, NH2), 4.27 (d, J = 4.3 Hz, H2'), 4.16 (d, J = 4.3 Hz, H3'), 4.06-4.03
(“m”, 1H, H4'), 3.94 (dd, J = 3.0, 11.5 Hz, 1H, H5'), 3.72 (dd, J = 2.1, 11.5 Hz, 1H, H5''),
0.91 (s, 9H, tBut), 0.84 (s, 9H, tBut), 0.74 (s, 9H, tBut), 0.10 (s, 3H, CH3), 0.09 (s, 3H,
123
CH3), 0.00 (s, 3H, CH3), 0.01 (s, 3H, CH3), -0.12 (s, 3H, CH3), -0.24 (s, 3H, CH3);
HRMS (ESI) m/z 745.4651 [M-CH3+H]+, calcd for C29H52IN5O4Si3 745.9249.
7-(1H-benzo[d][1,2,3]triazol-1-yl)tubercidin (144). Tert-butyl hydroperoxide (85 μL,
0.75 mmol) and iodine (38 mg, 0.15 mmol) were added to a stirred suspension of 19a
(100 mg, 0.38 mmol) and benzotriazole (89.5 mg, 0.75 mmol) in anhydrous DMF (2mL).
The resulting dark brown solution was stirred at 35 oC for 72 h, by which time TLC
showed ~10% conversion to a less polar product. A second portion of TBHP (85 μL, 0.75
mmol) was added and the resulting dark brown solution was stirred at 35 oC for 24 h
(total reaction time: 96 h). Volatiles were evaporated and co-evaporated with toluene
(high vacuum rotavap, <50 oC). The resulting brown gum was column chromatographed
(5% MeOH in EtOAc) to give 144 (26 mg, 18%) as a brown gum. This brown gum was
then injected into a semi-preparative HPLC column (Phenomenex Gemini RP-C18
column; 5µ, 25 cm x 1 cm) via a 5 mL loop and was eluted with an isocratic mobile
phase mixture 20% CH3CN in H2O at a flow rate of 1.5 mL/min to give 144 (tR = 21 min)
as a white powder: UV (MeOH) λmax 278 nm (ε = 16700), λmin 240 nm (ε = 8000); 1H
NMR (DMSO-d6) δ 8.50 (d, 1H, J = 8.3 Hz, Ph), 8.22 (s,1H, H2), 7.70-7.67 (m,1H, Ph),
7.62-7.52 (m, 4H, Ph, NH2), 7.04 (s, 1H, H8), 5.58 (dd, J=3.5,8.8, 3H, 5'-OH), 5.23 (d,
J=6.9 Hz, 1H, H1'), 5.20 (d, J=6.4 Hz, 1H,2'-OH), 4.92 (“dd”, J=6.4,11.2 Hz, 3'-OH,
H2'), 3.82 (“brs”, 1H, H3'), 3.75 (“d”, J=3.7, 6.2 Hz, H4'), 3.43 (dt, J=3.6,11.9 Hz, H5'),
3.28-3.22 (m, 1H, H5''); 13C NMR (DMSO-d6): δ 158.22, 153.13 (C2), 148.82, 144.52,
135.13, 129.42 (Ph), 125.63, 125.10 (Ph), 119.70 (Ph), 110.51 (Ph), 102.04, 99.48 (C8),
88.20 (C1'), 85.97 (C4'), 71.31 (C2'), 70.77 (C3'), 62.12 (C5'); HRMS (ESI): m/z
384.1391 [M+H]+, calcd for C17H17N7O4 384.1415.
124
5. CONCLUSION
In conclusion, I developed a novel desulfurization-difluorination method for the
synthesis of α,α-difluoro esters from the corresponding α-arylthio esters, wherein the
thiol group is present on the secondary internal carbon. 1,3-Dibromo-5,5-
dimethylhydantoin (DBH) was used as the oxidant (Br+) and Py.9HF was the fluoride ion
(F-) source. Arylthio esters with hydrogen as well as a ring deactivating chloro substituent
at the para position of the aromatic ring were converted to the corresponding gem-
difluoro esters under mild reaction conditions. However, the presence of a ring activating
methoxy substituent on the para position of the aromatic ring caused the reaction to be
sluggish and the difluorination was observed in low yields. Expansion of this
desulfurization-difluorination methodology towards the thioether substrates having
aldehyde, ketone, and unactivated alkane functional groups on the adjacent carbon was
unsuccessful. Also, treatment of lactones under similar reaction conditions gave low yield
conversion to the corresponding α-fluoro sulfides and α-fluoro sulfoxides. Attempted
synthesis of gem-difluorouridine derivatives from the corresponding 2'-arylthio uridine or
2'-fluoro-2'-arylthio uridine precursors was unsuccessful and resulted mostly in
halogenation of the pyrimidine ring at the C-5 and monofluorination at C2', without
desulfurization. However, treatment of the fully benzylated uridine substrate bearing a p-
methoxyphenylthioether substituent at the C2' with combination of NIS and DAST gave
a moderate but encouraging yield (44%) of the corresponding monofluorinated thioether
without any appreciable oxidation of thioether and iodination of the pyrimidine ring.
Chemical reduction protocols in combination with cyclic voltammetry were
employed for reductive desulfonylation of 2'-arylsulfonyl-2'-deoxyuridines as a possible
125
alternative process towards incorporation of fluorine and other substituents at the C2'. A
critical impact on the outcome of these reactions was the choice of the protecting groups.
Thus, 3',5'-O-diacetylated substrates 91a were found to decompose readily into the
corresponding vinyl sulfone 102 during silica gel column purification, whereas the fully
benzylated substrate 91b was prone to uracil elimination in the presence of strong base.
The cleavage of the C2'-S was found to occur at a relatively high cathodic potential close
to - 2.4 V vs SCE, with the expulsion of the corresponding sulfinate, meaning that strong
chemical reducing agents or selective electrochemical reductions were required.
Interestingly the protecting group (Bn or Ac) had no impact on the reduction potential.
Reactions of 2'-arylsulfonyl-2'-deoxy-2'-fluorouridines with using Organic Electron
Donors (OEDs) under different conditions (excess of reducing agents, temperature, uv
light activation) gave products resulting from the elimination of either acetate anion from
C3' position or uracil from C1' upon reduction of the C2'-S bond leading to furan-type
products. Interestingly, less powerful reducing-agent TDAE under UV light activation
was found to cleave the C2'-sulfonyl bond to give 2',3'-unsaturated 2'-fluorouridine (18%)
and 2'-fluoro-2'-deoxyuridine (10%) products. Tentative trapping of the resulting C2'-
anion under these uv light conditions either with Selectfluor or MeI were however
unsuccessful.
Bromination at C5-position of pyrimidine nucleosides such as uridine, 2'-
deoxyuiridine, arabinouridine, cytidine, and N4-benzoylcytidine were achieved under
mild reaction conditions using DBH. Also, transformation of purine nucleosides such as
adenosine, 2'-deoxuadenosine, guanosine, and 2'-deoxyguanosine to their corresponding
8-brominated counterparts with DBH. Although purine nucleosides required higher
126
amounts of reagents and produced brominated products in lower yields when compared
to that of the pyrimidine counterparts (47-98% vs 70-98%). Increase in temperature and
presence of Lewis acids such as TMSOTf and TsOH increased the rate of bromination
reaction and reduced the equivalents of DBH reagent required. The DBH bromination
protocol is bench friendly, with most conversions taking place at ambient temperature
and producing the brominated products in moderate to high yields. Also, acid labile
protection groups such as isopropylidine, and base labile protection groups such as acetyl,
benzoyl, and silyl were found to be stable during the course of bromination. In addition, I
developed novel NBS or DBH mediated bromination methodology for the synthesis of
previously known 8-bromotoyocamycin and 8-bromosangivamycin. This methodology
offers mild and improved synthetic alternative from previous reaction conditions. I also
found that DBH-bromination of various unprotected nucleosides proceeded efficiently in
MeOH, avoiding the need to use high-boiling and expensive solvent DMF.
Novel 6-N-benzyltubercidin and 6-N-(4-nitrobenzyl) analogues of tubercidin and
sangivamycin were synthesized by initial alkylation at N1 with respective benzyl
bromide followed by Me2NH mediated Dimroth rearrangement. However, no evidence of
formation of an initial N1-alkylated cationic intermediate was observed on TLC upon
treatment of toyocamycin with 4-nitrobenzyl bromide. Isolated less polar product
indicated direct alkylation on the exocyclic amino group to give 4-N-(4-
nitrobenzyl)toyocamycin. Alternatively, 6-N-(4-nitrobenzyl)tubercidin was also prepared
by (i) conversion of acetylated tubercidin to its 6-fluorocounterpart via Balz-Schiemann
type fluoro-diazotization with NaNO2/Py.9HF (55% HF); (ii) SNAr displacement of the
6-fluoro group by 4-nitrobenzylamine; (iii) deacetylation with NH3/MeOH. The 6-N-(4-
127
nitrobenzyl) derivatives of tubercidin and sangivamycin inhibited cross-membrane
transport of labelled uridine by the human equilibrative nucleoside transporter hENT1 at
>1 µM and ~120 nM, respectively. Inhibition of the proliferation of L1210, HeLa, and
PC-3 tumor cells in culture was observed with 6-N-benzyltubercidin and the 6-N-(4-
nitrobenzyl) derivatives of sangivamycin and toyocamycin at 0.92–9.4 µM
concentrations.
Novel 8-azidotoyocamycin was synthesized by treatment of 8-bromotoyocamycin
with sodium azide. But the azide product was light sensitive and decomposed after
exposure to light. Strain promoted click chemistry of 8-azidotoyocamycin with
cyclooctynes gave the corresponding 8-triazolyl derivatives in moderate yields. Iodine
mediated CH arylation of tubercidin with benzotriazole gave the corresponding 7-
substituted benzotriazolyl product, which showed emission at 420 nm with a quantum
yield of 0.002 s.
REFERENCES
(1) Levene, P. A.; Jacobs, W. A. Ber. Dtsch. Chem. Ges. 1909, 42, 1198.
(2) Avery, O. T.; MacLeod, C. M.; McCarty, M. J. Exp. Med. 1944, 79, 137.
(3) Watson, J. D.; Crick, F. H. C. Nature (London, U. K.) 1953, 171, 737.
(4) Parker, W. B. Chem. Rev. 2009, 109, 2880.
(5) Wang, P.; Chun, B.-K.; Rachakonda, S.; Du, J.; Khan, N.; Shi, J.; Stec, W.; Cleary, D.; Ross, B. S.; Sofia, M. J. J. Org. Chem. 2009, 74, 6819.
(6) Anderson, D. L. Drugs Today 2009, 45, 331.
(7) Mueller, K.; Faeh, C.; Diederich, F. Science 2007, 317, 1881.
(8) O'Hagan, D. J. Fluorine Chem. 2010, 131, 1071.
128
(9) Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Chem. Soc. Rev. 2008, 37, 320.
(10) Smart, B. E. Journal of Fluorine Chemistry 2001, 109, 3.
(11) Qiu, X.-L.; Xu, X.-H.; Qing, F.-L. Tetrahedron 2010, 66, 789.
(12) Hertel, L. W.; Kroin, J. S.; Misner, J. W.; Tustin, J. M. J. Org. Chem. 1988, 53, 2406.
(13) Gesto, D. S.; Cerqueira, N. M. F. S. A.; Fernandes, P. A.; Ramos, M. J. Curr. Med. Chem. 2012, 19, 1076.
(14) Montgomery, J. A.; Shortnacy-Fowler, A. T.; Clayton, S. D.; Riordan, J. M.; Secrist, J. A., III J. Med. Chem. 1992, 35, 397.
(15) Bonate, P. L.; Arthaud, L.; Cantrell, W. R., Jr.; Stephenson, K.; Secrist, J. A., III; Weitman, S. Nat. Rev. Drug Discovery 2006, 5, 855.
(16) Plunkett, W.; Huang, P.; Xu, Y.-Z.; Heinemann, V.; Grunewald, R.; Gandhi, V. Semin. Oncol. 1995, 22, 3.
(17) Manegold, C. Expert Rev. Anticancer Ther. 2004, 4, 345.
(18) Mini, E.; Nobili, S.; Caciagli, B.; Landini, I.; Mazzei, T. Ann Oncol 2006, 17 Suppl 5, v7.
(19) Elnaggar, M.; Giovannetti, E.; Peters, G. J. Curr. Pharm. Des. 2012, 18, 2811.
(20) de Sousa Cavalcante, L.; Monteiro, G. Eur. J. Pharmacol. 2014, 741, 8.
(21) Brown, K.; Dixey, M.; Weymouth-Wilson, A.; Linclau, B. Carbohydr. Res. 2014, 387, 59.
(22) Saischek, G.; Saischek, E. WO 2007/053869 A2, 2007.
(23) Heinemann, V.; Schulz, L.; Issels, R. D.; Plunkett, W. Semin. Oncol. 1995, 22, 11.
(24) Ruiz van Haperen, V. W. T.; Veerman, G.; Vermorken, J. B.; Peters, G. J. Biochem. Pharmacol. 1993, 46, 762.
(25) Heinemann, V.; Xu, Y. Z.; Chubb, S.; Sen, A.; Hertel, L. W.; Grindey, G. B.; Plunkett, W. Mol. Pharm. 1990, 38, 567.
(26) Plunkett, W.; Huang, P.; Xu, Y.-Z.; Heinemann, V.; Grunewald, R.; Gandhi, V. Semin. Oncol. 1995, 22, 3.
129
(27) Baker, C. H.; Banzon, J.; Bollinger, J. M.; Stubbe, J.; Samano, V.; Robins, M. J.; Lippert, B.; Jarvi, E.; Resvick, R. J. Med. Chem. 1991, 34, 1879.
(28) Silva, D. J.; Stubbe, J.; Samano, V.; Robins, M. J. Biochemistry 1998, 37, 5528.
(29) van der Donk, W. A.; Yu, G. X.; Perez, L.; Sanchez, R. J.; Stubbe, J.; Samano, V.; Robins, M. J. Biochemistry 1998, 37, 6419.
(30) Wang, J.; Lohman, G. J. S.; Stubbe, J. Biochemistry 2009, 48, 11612.
(31) Artin, E.; Wang, J.; Lohman, G. J. S.; Yokoyama, K.; Yu, G. X.; Griffin, R. G.; Bar, G.; Stubbe, J. Biochemistry 2009, 48, 11622.
(32) Eklund, H.; Uhlin, U.; Farnegardh, M.; Logan, D. T.; Nordlund, P. Prog Biophys Mol Bio 2001, 77, 177.
(33) Stubbe, J.; van der Donk, W. A. Chem. Biol. 1995, 2, 793.
(34) Wilkinson, J. A. Chemical Reviews 1992, 92, 505.
(35) Resnati, G. Tetrahedron 1993, 49, 9385.
(36) Lal, G. S.; Pez, G. P.; Syvret, R. G. Chemical Reviews 1996, 96, 1737.
(37) Tozer, M. J.; Herpin, T. F. Tetrahedron 1996, 52, 8619.
(38) Kirk, K. L. Organic Process Research & Development 2008, 12, 305.
(39) Kuroboshi, M.; Kanie, K.; Hiyama, T. Adv. Synth. Catal. 2001, 343, 235.
(40) Hugenberg, V.; Haufe, G. J. Fluorine Chem. 2012, 143, 238.
(41) Sondej, S. C.; Katzenellenbogen, J. A. J. Org. Chem. 1986, 51, 3508.
(42) Hugenberg, V.; Haufe, G. Synlett 2009, 106.
(43) Hugenberg, V.; Wagner, S.; Kopka, K.; Schober, O.; Schaefers, M.; Haufe, G. J. Org. Chem. 2010, 75, 6086.
(44) Ayuba, S.; Fukuhara, T.; Hara, S. Org. Lett. 2003, 5, 2873.
(45) Ayuba, S.; Hiramatsu, C.; Fukuhara, T.; Hara, S. Tetrahedron 2004, 60, 11445.
(46) Fukuhara, T.; Hara, S. Synlett 2009, 198.
(47) Fukuhara, T.; Hara, S. J. Org. Chem. 2010, 75, 7393.
130
(48) Ayuba, S.; Yoneda, N.; Fukuhara, T.; Hara, S. Bull. Chem. Soc. Jpn. 2002, 75, 1597.
(49) Hara, S.; Monoi, M.; Umemura, R.; Fuse, C. Tetrahedron 2012, 68, 10145.
(50) Shishimi, T.; Hara, S. J. Fluorine Chem. 2014, 168, 55.
(51) Srivastava, P. C.; Robins, R. K.; Meyer, R. B. In Chemistry of Nucleosides and Nucleotides; Townsend, L. B., Ed.; Plenum Press: New York, 1988; Vol. 1, p 113.
(52) Ueda, T. In Chemistry of Nucleosides and Nucleotides; Townsend, L. B., Ed.; Plenum Press: New York, 1988; Vol. 1, p 1.
(53) Agrofoglio, L. A.; Gillaizeau, I.; Saito, Y. Chem. Rev. 2003, 103, 1875.
(54) Clima, L.; Bannwarth, W. Helv. Chim. Acta 2008, 91, 165.
(55) Machida, H.; Sakata, S. In Nucleosides and Nucleotides as Antitumor and Antiviral Agents; Chu, C. K., Baker, D. C., Eds.; Plenum Press: New York, 1993, p 245.
(56) Robins, M. J.; Barr, P. J. J. Org. Chem. 1983, 48, 1854.
(57) De Clercq, E.; Descamps, J.; Balzarini, J.; Giziewicz, J.; Barr, P. J.; Robins, M. J. J. Med. Chem. 1983, 26, 661.
(58) McGuigan, C.; Yarnold, C. J.; Jones, G.; Velázquez, S.; Barucki, H.; Brancale, A.; Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J. J. Med. Chem. 1999, 42, 4479.
(59) Mercer, J. R.; Xu, L. H.; Knaus, E. E.; Wiebe, L. I. J. Med. Chem. 1989, 32, 1289.
(60) Beltz, R. E.; Visser, D. W. J. Am. Chem. Soc. 1955, 77, 736.
(61) Srivastava, P. C.; Nagpal, K. L. Experientia 1970, 26, 220.
(62) Kumar, V.; Yap, J.; Muroyama, A.; Malhotra, S. V. Synthesis 2009, 3957.
(63) Ryu, E. K.; MacCoss, M. J. J. Org. Chem. 1981, 46, 2819.
(64) Asakura, J.; Robins, M. J. J. Org. Chem. 1990, 55, 4928.
(65) Ross, S. A.; Burrows, C. J. Tetrahedron Lett. 1997, 38, 2805.
(66) Fukuhara, T. K.; Visser, D. W. J. Am. Chem. Soc. 1955, 77, 2393.
(67) Holmes, R. E.; Robins, R. K. J. Am. Chem. Soc. 1964, 86, 1242.
131
(68) Markish, I.; Arrad, O. Ind. Eng. Chem. Res. 1995, 34, 2125.
(69) Virgil, S. C. Encyclopedia of Reagents for Organic Synthesis 2001, DOI: 10.1002/047084289X.rd038.
(70) Alam, A. Synlett 2005, 2005, 2403.
(71) Auerbach, J.; Weissman, S. A.; Blacklock, T. J.; Angeles, M. R.; Hoogsteen, K. Tetrahedron Lett. 1993, 34, 931.
(72) Eguchi, H.; Kawaguchi, H.; Yoshinaga, S.; Nishida, A.; Nishiguchi, T.; Fujisaki, S. Bull. Chem. Soc. Jpn. 1994, 67, 1918.
(73) Herault, X.; Bovonsombat, P.; McNelis, E. Org. Prep. Proced. Int. 1995, 27, 652.
(74) Chassaing, C.; Haudrechy, A.; Langlois, Y. Tetrahedron Lett. 1997, 38, 4415.
(75) Alam, A.; Takaguchi, Y.; Ito, H.; Yushida, T.; Tsuboi, S. Tetrahedron 2005, 61, 1909.
(76) Shibatomi, K.; Zhang, Y.; Yamamoto, H. Chem. Asian J. 2008, 3, 1581
(77) Alam, A.; Takaguchi, Y.; Stuboi, S. Synth. Commun. 2005, 35, 1329.
(78) Khazaei, A.; Zolfigol, M. A.; Rostami, A. Synthesis 2004, 2959.
(79) Chen, Z.-Z.; Guan, X.-X.; Zheng, Z.-B.; Zou, X.-Z. J. East China Normal Univ.; Natural Science 2010, 7, 125.
(80) Suhadolnik, R. J. Nucleoside Antibiotics; Wiley-Interscience, 1970.
(81) Suhadolnik, R. J. Nucleosides as Biological Probes; Wiley, 1979.
(82) Seela, F.; Budow, S.; Peng, X. Curr. Org. Chem. 2012, 16, 161.
(83) Seela, F.; Peng, X.; Budow, S. Curr. Org. Chem. 2007, 11, 427.
(84) Seela, F.; Peng, X. Curr. Top. Med. Chem. (Sharjah, United Arab Emirates) 2006, 6, 867.
(85) Gerster, J. F.; Carpenter, B.; Robins, R. K.; Townsend, L. B. J. Med. Chem. 1967, 10, 326.
(86) Tolman, R. L.; Robins, R. K.; Townsend, L. B. J. Heterocycl. Chem. 1967, 4, 230.
(87) Tolman, R. L.; Robins, R. K.; Townsend, L. B. J. Amer. Chem. Soc. 1969, 91, 2102.
132
(88) Hinshaw, B. C.; Gerster, J. F.; Robins, R. K.; Townsend, L. B. J. Heterocycl. Chem. 1969, 6, 215.
(89) Uematsu, T.; Suhadolnik, R. J. J. Med. Chem. 1973, 16, 1405.
(90) Maruyama, T.; Wotring, L. L.; Townsend, L. B. J. Med. Chem. 1983, 26, 25.
(91) Bergstrom, D. E.; Brattesani, A. J.; Ogawa, M. K.; Reddy, P. A.; Schweickert, M. J.; Balzarini, J.; De Clercq, E. J. Med. Chem. 1984, 27, 285.
(92) De Clercq, E.; Balzarini, J.; Madej, D.; Hansske, F.; Robins, M. J. J. Med. Chem. 1987, 30, 481.
(93) Turk, S. R.; Shipman, C.; Nassiri, R.; Genzlinger, G.; Krawczyk, S. H.; Townsend, L. B.; Drach, J. C. Antimicrob. Agents Chemother. 1987, 31, 544.
(94) Gupta, P. K.; Daunert, S.; Nassiri, M. R.; Wotring, L. L.; Drach, J. C.; Townsend, L. B. J. Med. Chem. 1989, 32, 402.
(95) Robins, M. J.; Wilson, J. S.; Madej, D.; Low, N. H.; Hansske, F.; Wnuk, S. F. J. Org. Chem. 1995, 60, 7902.
(96) Bookser, B. C.; Matelich, M. C.; Ollis, K.; Ugarkar, B. G. J. Med. Chem. 2005, 48, 3389.
(97) Zhang, X.; Jia, D.; Liu, H.; Zhu, N.; Zhang, W.; Feng, J.; Yin, J.; Hao, B.; Cui, D.; Deng, Y.; Xie, D.; He, L.; Li, B. PLoS ONE 2013, 8, 1.
(98) Stockwin, L.; Yu, S.; Stotler, H.; Hollingshead, M.; Newton, D. BMC Cancer 2009, 9, 63.
(99) Choi, B. Y.; Lee, C.-H. Bioorg. Med. Chem. Lett. 2010, 20, 3880.
(100) Wu, R.; Smidansky, E. D.; Oh, H. S.; Takhampunya, R.; Padmanabhan, R.; Cameron, C. E.; Peterson, B. R. J. Med. Chem. 2010, 53, 7958.
(101) De Clercq, E.; Robins, M. J. Antimicrob. Agents Chemother. 1986, 30, 719.
(102) Varaprasad, C. V. N. S.; Ramasamy, K. S.; Girardet, J.-L.; Gunic, E.; Lai, V.; Zhong, W.; An, H.; Hong, Z. Bioorg. Chem. 2007, 35, 25.
(103) Ding, Y.; An, H.; Hong, Z.; Girardet, J.-L. Bioorg. Med. Chem. Lett. 2005, 15, 725.
(104) Eldrup, A. B.; Prhavc, M.; Brooks, J.; Bhat, B.; Prakash, T. P.; Song, Q.; Bera, S.; Bhat, N.; Dande, P.; Cook, P. D.; Bennett, C. F.; Carroll, S. S.; Ball, R. G.; Bosserman, M.; Burlein, C.; Colwell, L. F.; Fay, J. F.; Flores, O. A.; Getty, K.;
133
LaFemina, R. L.; Leone, J.; MacCoss, M.; McMasters, D. R.; Tomassini, J. E.; von Langen, D.; Wolanski, B.; Olsen, D. B. J. Med. Chem. 2004, 47, 5284.
(105) Olsen, D. B.; Eldrup, A. B.; Bartholomew, L.; Bhat, B.; Bosserman, M. R.; Ceccacci, A.; Colwell, L. F.; Fay, J. F.; Flores, O. A.; Getty, K. L.; Grobler, J. A.; LaFemina, R. L.; Markel, E. J.; Migliaccio, G.; Prhavc, M.; Stahlhut, M. W.; Tomassini, J. E.; MacCoss, M.; Hazuda, D. J.; Carroll, S. S. Antimicrob. Agents Chemother. 2004, 48, 3944.
(106) Bhattacharya, B. K.; Rao, T. S.; Revankar, G. R. J. Chem. Soc., Perkin Trans. 1 1995, 1543.
(107) Bhattacharya, B. K.; Ojwang, J. O.; Rando, R. F.; Huffman, J. H.; Revankar, G. R. J. Med. Chem. 1995, 38, 3957.
(108) Ojwang, J. O.; Bhattacharya, B. K.; Marshall, H. B.; Korba, B. E.; Revankar, G. R.; Rando, R. F. Antimicrob. Agents Chemother. 1995, 39, 2570.
(109) Ugarkar, B. G.; Castellino, A. J.; DaRe, J. M.; Kopcho, J. J.; Wiesner, J. B.; Schanzer, J. M.; Erion, M. D. J. Med. Chem. 2000, 43, 2894.
(110) Boyer, S. H.; Ugarkar, B. G.; Solbach, J.; Kopcho, J.; Matelich, M. C.; Ollis, K.; Gomez-Galeno, J. E.; Mendonca, R.; Tsuchiya, M.; Nagahisa, A.; Nakane, M.; Wiesner, J. B.; Erion, M. D. J. Med. Chem. 2005, 48, 6430.
(111) Naus, P.; Pohl, R.; Votruba, I.; Dzubak, P.; Hajduch, M.; Ameral, R.; Birkus, G.; Wang, T.; Ray, A. S.; Mackman, R.; Cihlar, T.; Hocek, M. J. Med. Chem. 2010, 53, 460.
(112) Bourderioux, A.; Naus, P.; Perlikova, P.; Pohl, R.; Pichova, I.; Votruba, I.; Dzubak, P.; Konecny, P.; Hajduch, M.; Stray, K. M.; Wang, T.; Ray, A. S.; Feng, J. Y.; Birkus, G.; Cihlar, T.; Hocek, M. J. Med. Chem. 2011, 54, 5498.
(113) Naus, P.; Caletkova, O.; Konecny, P.; Dzubak, P.; Bogdanova, K.; Kolar, M.; Vrbkova, J.; Slavetinska, L.; Tloust'ova, E.; Perlikova, P.; Hajduch, M.; Hocek, M. J. Med. Chem. 2014, 57, 1097.
(114) Miles, R. W.; Samano, V.; Robins, M. J. J. Am. Chem. Soc. 1995, 117, 5951.
(115) Robins, M. J.; Trip, E. M. Biochemistry 1973, 12, 2179.
(116) Young, J. D.; Yao, S. Y. M.; Baldwin, J. M.; Cass, C. E.; Baldwin, S. A. Mol. Aspects Med. 2013, 34, 529.
(117) Jordheim, L. P.; Durantel, D.; Zoulim, F.; Dumontet, C. Nat. Rev. Drug Discov. 2013, 12, 447.
134
(118) Griffiths, M.; Beaumont, N.; Yao, S. Y. M.; Sundaram, M.; Boumah, C. E.; Davies, A.; Kwong, F. Y. P.; Coe, I.; Cass, C. E.; Young, J. D.; Baldwin, S. A. Nat. Med. 1997, 3, 89.
(119) Robins, M. J.; Asakura, J.-i.; Kaneko, M.; Shibuya, S.; Jakobs, E. S.; Agbanyo, F. R.; Cass, C. E.; Paterson, A. R. P. Nucleosides and Nucleotides 1994, 13, 1627.
(120) Robins, M. J.; Peng, Y.; Damaraju, V. L.; Mowles, D.; Barron, G.; Tackaberry, T.; Young, J. D.; Cass, C. E. J. Med. Chem. 2010, 53, 6040.
(121) Young, J. D.; Yao, S. Y. M.; Cass, C. E.; Baldwin, S. A.; : Red Cell Membrane Transport in Health and Disease, Bernhardt, I., Ellory, J. C., (Eds.), Springer-Verlag, Berlin: 2003, p 321.
(122) Hugenberg, V.; Wagner, S.; Kopka, K.; Schober, O.; Schafers, M.; Haufe, G. J Org Chem 2010, 75, 6086.
(123) Unsuccessful attempt of bromination of inosine with NBS in DMF has been reported.61
(124) Tolman, R. L.; Robins, R. K.; Townsend, L. B. J. Amer. Chem. Soc. 1969, 91, 2102.
(125) Tolman, R. L.; Robins, R. K.; Townsend, L. B. J. Heterocycl. Chem. 1971, 8, 703.
(126) Chassaing, C.; Haudrechy, A.; Langlois, Y. Tetrahedron Lett. 1997, 38, 4415.
(127) Biswas, S.; Dahlstrand, C.; Watile, R. A.; Kalek, M.; Himo, F.; Samec, J. S. M. Chem. Eur. J. 2013, 19, 17939.
(128) Aveniente, M.; Pinto, E. F.; Santos, L. S.; Rossi-Bergmann, B.; Barata, L. E. S. Bioorg. Med. Chem. 2007, 15, 7337.
(129) Kato, M.; Ouchi, A.; Yoshikoshi, A. Bull. Chem. Soc. Jpn. 1991, 64, 1479.
(130) Moens, M.; Verniest, G.; De Schrijver, M.; ten Holte, P.; Thuring, J.-W.; Deroose, F.; De Kimpe, N. Tetrahedron 2012, 68, 9284.
(131) Tomioka, K.; Ishiguro, T.; Iitaka, Y.; Koga, K. Tetrahedron 1984, 40, 1303.
(132) Hampton, A.; Nichol, A. W. Biochemistry 1966, 5, 2076.
(133) Matsuda, A.; Miyasaka, T. Heterocycles 1983, 20, 55.
(134) Robins, M. J.; Mullah, K. B.; Wnuk, S. F.; Dalley, N. K. J. Org. Chem. 1992, 57, 2357.
135
(135) McCarthy, J. R.; Peet, N. P.; LeTourneau, M. E.; Inbasekaran, M. J. Am. Chem. Soc. 1985, 107, 735.
(136) Wnuk, S. F.; Robins, M. J. J. Org. Chem. 1990, 55, 4757.
(137) Rayala, R.; Wnuk, S. F. Tetrahedron Lett. 2012, 53, 3333.
(138) Xu, B.; Unione, L.; Sardinha, J.; Wu, S.; Ethève-Quelquejeu, M.; Pilar Rauter, A.; Blériot, Y.; Zhang, Y.; Martín-Santamaría, S.; Díaz, D.; Jiménez-Barbero, J.; Sollogoub, M. Angew. Chem. Int. Ed. 2014, 53, 9597.
(139) Chen, H.; Hu, Z.; Zhang, J.; Liang, G.; Xu, B. Tetrahedron 2015, 71, 2089.
(140) Lebouc, A.; Martigny, P.; Carlier, R.; Simonet, J. Tetrahedron 1985, 41, 1251.
(141) Wnuk, S. F.; Robins, M. J. J. Am. Chem. Soc. 1996, 118, 2519.
(142) Najera, C.; Yus, M. Tetrahedron 1999, 55, 10547.
(143) Wnuk, S. F.; Rios, J. M.; Khan, J.; Hsu, Y.-L. J. Org. Chem. 2000, 65, 4169.
(144) Schoenebeck, F.; Murphy, J. A.; Zhou, S.-Z.; Uenoyama, Y.; Miclo, Y.; Tuttle, T. J. Am. Chem. Soc. 2007, 129, 13368.
(145) Coeffard, V.; Thobie-Gautier, T.; Beaudet, I.; Le Grognec, E.; Quintard, J.-P. Eur. J. Org. Chem. 2008, 383.
(146) Senboku, H.; Nakahara, K.; Fukuhara, T.; Hara, S. Tetrahedron Lett. 2010, 51, 435.
(147) Nielsen, M.; Jacobsen, C. B.; Holub, N.; Paixao, M. W.; Joergensen, K. A. Angew. Chem., Int. Ed. 2010, 49, 2668.
(148) Viaud, P.; Coeffard, V.; Thobie-Gautier, C.; Beaudet, I.; Galland, N.; Quintard, J.-P.; Le Grognec, E. Org. Lett. 2012, 14, 942.
(149) Xuan, J.; Li, B.-J.; Feng, Z.-J.; Sun, G.-D.; Ma, H.-H.; Yuan, Z.-W.; Chen, J.-R.; Lu, L.-Q.; Xiao, W.-J. Chem. - Asian J. 2013, 8, 1090.
(150) Yang, D.-T.; Meng, Q.-Y.; Zhong, J.-J.; Xiang, M.; Liu, Q.; Wu, L.-Z. Eur. J. Org. Chem. 2013, 2013, 7528.
(151) Clive, D. L. J.; Wickens, P. L.; Sgarbi, P. W. M. The Journal of Organic Chemistry 1996, 61, 7426.
(152) Robins, M. J.; Lewandowska, E.; Wnuk, S. F. J. Org. Chem. 1998, 63, 7375.
136
(153) Robins, M. J.; Wilson, J. S.; Madej, D.; Low, N. H.; Hansske, F.; Wnuk, S. F. J. Org. Chem. 1995, 60, 7902.
(154) Mengel, R.; Seifert, J. M. Tetrahedron Lett. 1977, 4203.
(155) Adachi, T.; Iwasaki, T.; Inoue, I.; Miyoshi, M. J. Org. Chem. 1979, 44, 1404.
(156) Amino, Y.; Iwagami, H. Chem. Pharm. Bull. 1991, 39, 622.
(157) Johansen, O.; Holan, G.; Marcuccio, S. M.; Mau, A. W. H. Aust. J. Chem. 1991, 44, 37.
(158) Manchand, P. S.; Belica, P. S.; Holman, M. J.; Huang, T. N.; Maehr, H.; Tam, S. Y. K.; Yang, R. T. J. Org. Chem. 1992, 57, 3473.
(159) Johansen, O.; Marcuccio, S. M.; Mau, A. W. H. Aust. J. Chem. 1994, 47, 1843.
(160) Guo, Z.; Sanghvi, Y. S.; Brammer, L. E., Jr.; Hudlicky, T. Nucleosides, Nucleotides Nucleic Acids 2001, 20, 1263.
(161) Liu, J.; Zhuang, S.; Gui, Q.; Chen, X.; Yang, Z.; Tan, Z. European Journal of Organic Chemistry 2014, 2014, 3196.
(162) Tong, W.; Xi, Z.; Gioeli, C.; Chattopadhyaya, J. Tetrahedron 1991, 47, 3431.
(163) Hoffmann, R. W. Angew. Chem. 1968, 80, 823.
(164) Hoffmann, R. W. Angew. Chem. Int. Ed. Engl. 1968, 7, 754.
(165) Wiberg, N. Angew. Chem. 1968, 80, 809.
(166) Wiberg, N. Angew. Chem. Int. Ed. Engl. 1968, 7, 766.
(167) Zhou, S.; Farwaha, H.; Murphy, J. A. Chimia 2012, 66, 418.
(168) Murphy, J. A. J. Org. Chem. 2014, 79, 3731.
(169) Doni, E.; Murphy, J. A. Chem. Commun. (Cambridge, U. K.) 2014, 50, 6073.
(170) Broggi, J.; Terme, T.; Vanelle, P. Angew. Chem., Int. Ed. 2014, 53, 384.
(171) Murphy, J. A.; Garnier, J.; Park, S. R.; Schoenebeck, F.; Zhou, S.-z.; Turner, A. T. Org. Lett. 2008, 10, 1227.
(172) In view of the high reduction potential of the arylsulfone moiety compared to OED, even transferring one electron would theoretically appear difficult. Nevertheless, it is well known that electron transfer through a mediator, such as a charge-transfer complex, in solution is frequently achieved more easily (i.e. at a
137
less negative potential) than would be expected from the bare electrochemical data (explained by the formation of intimate complexes and ion pairing).
(173) Martin, J. A.; Bushnell, D. J.; Duncan, I. B.; Dunsdon, S. J.; Hall, M. J.; Machin, P. J.; Merrett, J. H.; Parkes, K. E. B.; Roberts, N. A.; et, a. J. Med. Chem. 1990, 33, 2137.
(174) Robins, M. J.; Guo, Z.; Samano, M. C.; Wnuk, S. F. J. Am. Chem. Soc. 1999, 121, 1425.
(175) Dang, T. P.; Sobczak, A. J.; Mebel, A. M.; Chatgilialoglu, C.; Wnuk, S. F. Tetrahedron 2012, 68, 5655.
(176) Fritscher, J.; Artin, E.; Wnuk, S.; Bar, G.; Robblee, J. H.; Kacprzak, S.; Kaupp, M.; Griffin, R. G.; Bennati, M.; Stubbe, J. J. Am. Chem. Soc. 2005, 127, 7729.
(177) Wnuk, S. F.; Chowdhury, S. M.; Garcia, P. I., Jr.; Robins, M. J. J. Org. Chem. 2002, 67, 1816.
(178) Zipse, H.; Artin, E.; Wnuk, S.; Lohman, G. J. S.; Martino, D.; Griffin, R. G.; Kacprzak, S.; Kaupp, M.; Hoffman, B.; Bennati, M.; Stubbe, J.; Lees, N. J. Am. Chem. Soc. 2009, 131, 200.
(179) Adhikary, A.; Kumar, A.; Heizer, A. N.; Palmer, B. J.; Pottiboyina, V.; Liang, Y.; Wnuk, S. F.; Sevilla, M. D. J. Am. Chem. Soc. 2013, 135, 3121.
(180) Close, D. M.; Bernhard, W. A. J. Chem. Phys. 1979, 70, 210.
(181) Clark, J. L.; Hollecker, L.; Mason, J. C.; Stuyver, L. J.; Tharnish, P. M.; Lostia, S.; McBrayer, T. R.; Schinazi, R. F.; Watanabe, K. A.; Otto, M. J.; Furman, P. A.; Stec, W. J.; Patterson, S. E.; Pankiewicz, K. W. J. Med. Chem. 2005, 48, 5504.
(182) Adhikary, A.; Kumar, A.; Rayala, R.; Hindi, R. M.; Adhikary, A.; Wnuk, S. F.; Sevilla, M. D. J. Am. Chem. Soc. 2014, 136, 15646.
(183) Dimroth, O. Justus Liebigs Ann. Chem. 1909, 364, 183.
(184) Engel, J. D. Biochem. Biophys. Res. Commun. 1975, 64, 581.
(185) Fujii, T.; Itaya, T. Heterocycles 1998, 48, 359.
(186) Pande, P.; Shearer, J.; Yang, J.; Greenberg, W. A.; Rokita, S. E. J. Am. Chem. Soc. 1999, 121, 6773.
(187) Zhong, M.; Robins, M. J. J. Org. Chem. 2006, 71, 8901.
(188) Robins, M. J.; Uznański, B. Can. J. Chem. 1981, 59, 2608.
138
(189) Liu, J.; Robins, M. J. Org. Lett. 2005, 7, 1149.
(190) Liu, J.; Robins, M. J. J. Am. Chem. Soc. 2007, 129, 5962.
(191) Olah, G. A.; Welch, J. T.; Vankar, Y. D.; Nojima, M.; Kerekes, I.; Olah, J. A. J. Org. Chem. 1979, 44, 3872.
(192) Bookser, B. C.; Ugarkar, B. G.; Matelich, M. C.; Lemus, R. H.; Allan, M.; Tsuchiya, M.; Nakane, M.; Nagahisa, A.; Wiesner, J. B.; Erion, M. D. J. Med. Chem. 2005, 48, 7808.
(193) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 16793.
(194) Lutz, J.-F. Angew. Chem., Int. Ed. 2008, 47, 2182.
(195) Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. Science (Washington, DC, U. S.) 2008, 320, 664.
(196) Campbell-Verduyn, L. S.; Mirfeizi, L.; Schoonen, A. K.; Dierckx, R. A.; Elsinga, P. H.; Feringa, B. L. Angew. Chem., Int. Ed. 2011, 50, 11117.
(197) Marks, I. S.; Kang, J. S.; Jones, B. T.; Landmark, K. J.; Cleland, A. J.; Taton, T. A. Bioconjugate Chem. 2011, 22, 1259.
(198) Koo, H.; Lee, S.; Na, J. H.; Kim, S. H.; Hahn, S. K.; Choi, K.; Kwon, I. C.; Jeong, S. Y.; Kim, K. Angew. Chem., Int. Ed. 2012, 51, 11836.
(199) Heuer-Jungemann, A.; Kirkwood, R.; El-Sagheer, A. H.; Brown, T.; Kanaras, A. G. Nanoscale 2013, 5, 7209.
(200) Bergstrom, D. E.; Brattesani, A. J. Nucleic Acids Res. 1980, 8, 6213.
(201) Sawangphon, T.; Katrun, P.; Chaisiwamongkhol, K.; Pohmakotr, M.; Reutrakul, V.; Jaipetch, T.; Soorukram, D.; Kuhakarn, C. Synth. Commun. 2013, 43, 1692.
(202) Li, X.; Shi, X.; Fang, M.; Xu, X. J. Org. Chem. 2013, 78, 9499.
(203) Gao, Y.; Wu, W.; Huang, Y.; Huang, K.; Jiang, H. Org. Chem. Front. 2014, 1, 361.
(204) Lu, Q.; Zhang, J.; Zhao, G.; Qi, Y.; Wang, H.; Lei, A. J. Am. Chem. Soc. 2013, 135, 11481.
(205) Liu, Z.; Liao, P.; Bi, X. Org. Lett. 2014, 16, 3668.
139
(206) Liang, Y.; Wnuk, S. Molecules 2015, 20, 4874.
(207) Beukeaw, D.; Udomsasporn, K.; Yotphan, S. J. Org. Chem. 2015, 80, 3447.
(208) Saladino, R.; Mezzetti, M.; Mincione, E.; Palamara, A. T.; Savini, P.; Marini, S. Nucleosides Nucleotides 1999, 18, 2499.
(209) Kumar, R.; Wiebe, L. I.; Knaus, E. E. Can. J. Chem. 1994, 72, 2005.
(210) Kotra, L.; Pai, E. F.; Paige, C. J.; Bello, A. M. WO 2008/083465 A1, 2008
(211) Lin, T. S.; Cheng, J. C.; Ishiguro, K.; Sartorelli, A. C. J. Med. Chem. 1985, 28, 1481.
(212) Niles, J. C.; Wishnok, J. S.; Tannenbaum, S. R. Chem. Res. Toxicol. 2000, 13, 390.
(213) Lin, T. S.; Cheng, J. C.; Ishiguro, K.; Sartorelli, A. C. J. Med. Chem. 1985, 28, 1194.
(214) Lin, T. S.; Cheng, J. C.; Ishiguro, K.; Sartorelli, A. C. J. Med. Chem. 1985, 28, 1194.
(215) Gillet, L. C. J.; Schaerer, O. D. Org. Lett. 2002, 4, 4205.
(216) Watanabe, M.; Ezoe, Y.; Isozaki, M.; Kasahara, T.; Hayashi, S.; Tamamura, K. Proc. Sch. Sci. Tokai Univ. 1995, 30, 143.
(217) Lau, J.; Kodra, J. T.; Guzel, M.; Santosh, K. C.; Mjalli, A. M. M.; Andrews, R. C.; Polisetti, D. R.; Novo Nordisk A/S, Den. . 2003, p 175 pp.
(218) Aranapakam, V.; Grosu, G. T.; Davis, J. M.; Hu, B.; Ellingboe, J.; Baker, J. L.; Skotnicki, J. S.; Zask, A.; DiJoseph, J. F.; Sung, A.; Sharr, M. A.; Killar, L. M.; Walter, T.; Jin, G.; Cowling, R. J. Med. Chem. 2003, 46, 2361.
(219) Yang, Z. Y.; Burton, D. J. J. Org. Chem. 1992, 57, 5144.
(220) Hampton, A.; Nichol, A. W. Biochemistry 1966, 5, 2076.
(221) Mulchande, J.; Martins, L.; Moreira, R.; Archer, M.; Oliveira, T. F.; Iley, J. Org. Biomol. Chem. 2007, 5, 2617.
(222) Nowak, I.; Conda-Sheridan, M.; Robins, M. J. J. Org. Chem. 2005, 70, 7455.
(223) Chung, F.-L.; Earl, R. A.; Townsend, L. B. J. Org. Chem. 1980, 45, 4056.
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141
VITA
RAMANJANEYULU RAYALA
2002 - 2005 B.Sc., Chemistry
Kakatiya University, Warangal, India
2006 - 2008 M.Sc., Chemistry
Osmania University, Hyderabad, India
2008 – 2010 Chemistry Lecturer
KLR Degree College, Palwancha, india
2010 - 2015 Doctoral Candidate
Florida International University, Miami, Florida, USA
2011-12 “Outstanding Organic Chemistry Teaching Assistant” Award
2014-2015 Dissertation Year Fellowship award
American Chemical Society
PUBLICATIONS AND PRESENTATIONS
Adhikary, Amitava; Kumar, Anil; Rayala, Ramanjaneyulu; Hindi, Ragda M.; Adhikary, Ananya; Wnuk, Stanislaw F.; and Sevilla, Michael D., “One-electron oxidation of Gemcitabine and analogs: Mechanism of formation of C3′ and C2′ sugar radicals”, J. Am. Chem. Soc. 2014, 136 (44), 15646−15653.
Rayala, Ramanjaneyulu; Theard, Patricia; Ortiz, Heysell; Yao, Sylvia; Young, James D.; Balzarini, Jan; Robins, Morris J., Wnuk, Stanislaw F., “Synthesis of Purine and 7-Deazapurine Nucleoside Analogues of 6-N-(4-Nitrobenzyl)adenosine; Inhibition of Nucleoside Transport and Proliferation of Cancer Cells”, ChemMedChem 2014, 9, 2186-2192.
Rayala, Ramanjaneyulu; Wnuk, Stanislaw F., “Bromination at C-5 of pyrimidine and C-8 of purine nucleosides with 1,3-dibromo-5,5-dimethylhydantoin”, Tetrahedron Letters 2012, 53 (26), 3333-3336.
Rayala, Ramanjaneyulu; Theard, Patricia; Ortiz, Heysell; Wnuk, Stanislaw F.; Robins, Morris J., “Synthesis of 6-N-substituted 7-deazapurine nucleoside antibiotics: Potential
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nucleoside transport inhibitors”, 247th ACS National Meeting & Exposition, Dallas, TX, United States, March 16-20, 2014.
Rayala, Ramanjaneyulu; Giuglio, Gamal; Broggi, Julie; Medebielle, Maurice; Wnuk, Stanislaw F., “Reductive desulfonylation of 2'-(arylsulfonyl)-2'-deoxy-2'-fluorouridine derivatives with tetrakis(dimethylamino)ethylene”, 245th ACS National Meeting & Exposition, New Orleans, LA, United States, April 7-11, 2013.
Rayala, Ramanjaneyulu; Wnuk, Stanislaw F., “Bromination at C-5 of pyrimidine and C-8 of purine nucleosides with 1,3-dibromo-5,5-dimethylhydantoin”, 243rd ACS National Meeting & Exposition, San Diego, CA, United States, March 25-29, 2012.
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